Methods and systems for scaffolds comprising nanoelectronic components

ABSTRACT

The present invention generally relates to nanoscale wires and tissue engineering. Systems and methods are provided in various embodiments for preparing cell scaffolds that can be used for growing cells or tissues, where the cell scaffolds comprise nanoscale wires. In some cases, the nanoscale wires can be connected to electronic circuits extending externally of the cell scaffold. Such cell scaffolds can be used to grow cells or tissues which can be determined and/or controlled at very high resolutions, due to the presence of the nanoscale wires, and such cell scaffolds will find use in a wide variety of novel applications, including applications in tissue engineering, prosthetics, pacemakers, implants, or the like. This approach thus allows for the creation of fundamentally new types of functionalized cells and tissues, due to the high degree of electronic control offered by the nanoscale wires and electronic circuits.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/018,075, filed Sep. 4, 2013, entitled “Methods And Systems ForScaffolds Comprising Nanoelectronic Components,” by Lieber, et al.,which claims the benefit of U.S. Provisional Patent Application Ser. No.61/698,492, filed Sep. 7, 2012, entitled “Methods And Systems ForScaffolds Comprising Nanoelectronic Components,” by Lieber, et al., andof U.S. Provisional Patent Application Ser. No. 61/723,213, filed Nov.6, 2012, entitled “Methods And Systems For Scaffolds ComprisingNanoelectronic Components,” by Lieber, et al., each incorporated hereinby reference in its entirety.

GOVERNMENT FUNDING

Research leading to various aspects of the present invention wassponsored, at least in part, by the NIH, Grant No. 5DP1OD003900-04. TheU.S. Government has certain rights in the invention.

FIELD

The present invention generally relates to nanoscale wires and tissueengineering.

BACKGROUND

Recent efforts in coupling electronics and tissues have focused onflexible, stretchable planar arrays that conform to tissue surfaces, orimplantable microfabricated probes. These approaches have been limitedin merging electronics with tissues while minimizing tissue disruption,because the support structures and electronic detectors are generally ofa much larger scale than the extracellular matrix and the cells.Furthermore, planar arrays only probe near the tissue surface and cannotbe used to study the internal 3-dimensional structure of the tissue. Forexample, probes using nanowire field-effect transistors have shown thatelectronic devices with nanoscopic features can be used to detect extra-and intracellular potentials from single cells, but are limited to onlysurface recording from 3-dimensional tissues and organs. Accordingly,improvements in merging electronics with tissues are still needed.

SUMMARY

The present invention generally relates to nanoscale wires and tissueengineering. The subject matter of the present invention involves, insome cases, interrelated products, alternative solutions to a particularproblem, and/or a plurality of different uses of one or more systemsand/or articles.

In one aspect, the present invention is generally directed to a method.In one set of embodiments, the method includes acts of forming apolymeric material on at least a portion of a substrate, forming one ormore metal leads on the polymeric material, and removing at least aportion of the sacrificial material from the polymeric material. In somecases, the substrate comprises a sacrificial material. In certaininstances, the polymeric material comprises one or more nanoscale wires.

In another set of embodiments, the method includes acts of forming oneor more polymeric constructs on a substrate using e-beam lithography,removing at least a portion the substrate from the one or more polymericconstructs, and rolling the one or more polymeric constructs into a3-dimensional structure.

The method, in yet another set of embodiments, includes acts of formingone or more pre-stressed polymeric constructs on a substrate usinglithography, and removing the substrate from the one or morepre-stressed polymeric constructs. In some embodiments, removal of thesubstrate releases stress on the pre-stressed polymeric constructs suchthat the polymeric constructs form a 3-dimensional structure.

In another set of embodiments, the article comprises a biological tissuecomprising a semiconductor nanowire.

In some embodiments, the article comprises a tissue comprising nanoscalewires and metal leads extending between at least some of the nanoscalewires and a surface of the tissue. The article, in yet another set ofembodiments, includes a biological tissue comprising at least one kinkednanoscale wire. According to still another set of embodiments, thearticle comprises a biological tissue comprising a curled 2-dimensionalelectrical network therein.

The article, in accordance with one set of embodiments, includes abiological tissue comprising a cell scaffold. In some cases, at least aportion of the cell scaffold defines at least a portion of an electricalcircuit that extends externally of the cell scaffold.

According to another set of embodiments, the article includes abiological tissue comprising a cell scaffold comprising a plurality ofelectrical component responsive to an electrical property of thebiological tissue. In another set of embodiments, the article includes abiological tissue comprising a cell scaffold comprising a plurality ofpH-sensitive electrical components.

The article, in one set of embodiments, comprises a cell scaffoldcomprising one or more semiconductor nanowires and one or more polymericconstructs.

In another set of embodiments, the article comprises a cell scaffoldcomprising one or more nanoscale wires and one or more polymericconstructs. In some cases, at least one of the polymeric constructscontains a conductive pathway extending between a nanoscale wire and asurface of the cell scaffold.

According to another set of embodiments, the article comprises a cellscaffold comprising a field effect transistor and one or more polymericconstructs. In yet another set of embodiments, the article includes acell scaffold comprising at least one kinked nanoscale wire and one ormore polymeric constructs. In still another set of embodiments, thearticle includes a cell scaffold comprising a pH-sensitive nanoscalewire and one or more polymeric constructs. The article, in yet anotherset of embodiments, includes a cell scaffold comprising a sensorresponsive to an electrical property and one or more polymericconstructs, wherein the sensor comprises a nanoscale wire.

In one set of embodiments, the article comprises an electrical circuitdefined by one or more metal leads having a maximum cross-sectionaldimension of less than about 5 micrometers, forming a 3-dimensionalstructure having an average pore size of between about 100 micrometersand about 1.5 mm. The electrical circuit may comprise one or morenanoscale wires in certain embodiments.

In another set of embodiments, the article comprises an electricalcircuit defined by one or more metal leads, forming a 3-dimensionalstructure. In some cases, the electrical circuit comprises one or morepH-sensitive components.

Yet another set of embodiments is directed to an article that includesan electrical circuit defined by one or more metal leads, forming a3-dimensional structure having an open porosity of at least about 50%and an average pore size of between about 100 micrometers and about 1.5mm. In some instances, the electrical circuit comprises an electricalcomponent responsive to an electrical property external to theelectrical component.

In another set of embodiments, the method comprises acts of forming oneor more polymers on a substrate, removing at least a portion of thesubstrate from the one or more polymers, and forming the polymers andmetal leads into a 3-dimensional structure having an open porosity of atleast about 50% and an average pore size of between about 100micrometers and about 1.5 mm. In some cases, at least some of thepolymers may comprise metal leads.

The method, in yet another set of embodiments, includes acts of formingone or more polymers on a substrate, and removing at least a portion ofthe substrate from the one or more polymers. In some embodiments, theone or more polymers comprise semiconductor nanowires.

Still another set of embodiments is generally directed to a methodcomprising depositing nanoscale wires on a polymer positioned on asubstrate, and removing at least a portion of the substrate from thepolymer. In yet another set of embodiments, the method includes acts ofdepositing one or more kinked nanoscale wires on a polymer, and formingat least a portion of the polymer into a 3-dimensional structure havingan open porosity of at least about 50%.

According to one set of embodiments, the method includes an act ofdetermining pH of a biological tissue at a resolution of less than about1 mm. In another set of embodiments, the method includes an act ofdetermining an electrical property of a biological tissue in threedimensions using a field effect transistor disposed within thebiological tissue.

In another aspect, the present invention encompasses methods of makingone or more of the embodiments described herein, for example, cellscaffolds comprising nanoscale wires. In still another aspect, thepresent invention encompasses methods of using one or more of theembodiments described herein, for example, cell scaffolds comprisingnanoscale wires.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1C illustrate certain embodiments of the invention generallydirected to cell scaffolds comprising nanoscale wires;

FIG. 2 schematically illustrates a cross-section of one embodiment ofthe invention;

FIG. 3 is a schematic representation of an integrated system usingdiscrete building blocks of electronic and biological systems, in someembodiments of the invention;

FIGS. 4A-4I illustrate cell scaffolds comprising nanoscale wires, inaccordance with certain embodiments of the invention;

FIG. 5 illustrates the 3-dimensional distribution of nanoscale wires ina cell scaffold in yet another embodiment of the invention;

FIGS. 6A-6F illustrate the geometry of nanoscale wires, in accordancewith certain embodiments of the invention;

FIGS. 7A-7E illustrate design and fabrication of nanoscale wires, inaccordance with certain embodiments of the invention;

FIGS. 8A-8D illustrate cell scaffolds comprising nanoscale wires andanalysis of sensitivity of nanoscale wires over time, in accordance withcertain embodiments of the invention;

FIG. 9A-9D illustrate 3-dimensional culturing of neuronal cells in acell scaffold, in another embodiment of the invention;

FIG. 10A-10H illustrate 3-dimensional reconstructed confocalfluorescence image of a cell scaffold and analysis of toxicity andconductance of certain cell scaffolds in accordance with variousembodiments of the invention;

FIGS. 11A-11B illustrate 3-dimensional reconstructed confocalfluorescence image of a cell scaffold in accordance with one embodimentof the invention, illustrating the distribution of cells and metalswithin the scaffold;

FIGS. 12A-12H illustrate 3-dimensional culturing of cardiomyocytes in acell scaffold, in another embodiment of the invention;

FIGS. 13A-13C illustrate fluorescence images of cardiomyocytes growingon a cell scaffold in accordance with one embodiment of the invention;

FIGS. 14A-14C illustrate epi-fluorescence images of cardiomyocytesgrowing on a cell scaffold in accordance with one embodiment of theinvention;

FIGS. 15A-15C illustrate multiplexed electrical recordings of cellscaffolds comprising nanoscale wires, in accordance with certainembodiments of the invention;

FIGS. 16A-16C illustrate multiplexed 3D electrical recordings of cellscaffolds comprising nanoscale wires, in accordance with certainembodiments of the invention;

FIGS. 17A-17F illustrate a cell scaffold used as a vascular construct,in accordance with another embodiment of the invention.

FIGS. 18A-18H schematically illustrate growing cells on a vascular cellscaffold, in another embodiment of the invention.

FIG. 19 illustrates a confocal fluorescence image of smooth muscle cellsin a cell scaffold, in another embodiment of the invention.

FIGS. 20A-20H illustrate certain techniques for forming a cell scaffoldin one embodiment of the invention;

FIGS. 21A-21I illustrate certain techniques for forming a cell scaffoldin one embodiment of the invention.

DETAILED DESCRIPTION

The present invention generally relates to nanoscale wires and tissueengineering. Systems and methods are provided in various embodiments forpreparing cell scaffolds that can be used for growing cells or tissues,where the cell scaffolds comprise nanoscale wires. In some cases, thenanoscale wires can be connected to electronic circuits extendingexternally of the cell scaffold. Such cell scaffolds can be used to growcells or tissues which can be determined and/or controlled at very highresolutions, due to the presence of the nanoscale wires, and such cellscaffolds will find use in a wide variety of novel applications,including applications in tissue engineering, prosthetics, pacemakers,implants, or the like. This approach thus allows for the creation offundamentally new types of functionalized cells and tissues, due to thehigh degree of electronic control offered by the nanoscale wires andelectronic circuits.

For example, certain aspects of the present invention are generallydirected to cell scaffolds comprising nanoscale wires and one or morepolymeric constructs. The cell scaffolds can be fabricated, for example,using well-known lithographic techniques such as those discussed herein.The cell scaffolds may also comprise metal leads that create conductivepathways between the nanoscale wires and the surface of the cellscaffold. Accordingly, the electrical properties of the nanoscale wires,and any surrounding cells or tissue grown on the cell scaffolds, can bedetermined and/or controlled, e.g., externally of the cell scaffolds.While others may have previously suggested using nanoscale wires in cellscaffolding materials, such applications have never involvedincorporating the nanoscale wires in electronic circuits, let aloneusing the electronic circuits to determine or control the nanoscalewires.

Turning first to FIG. 1A, a representative cell scaffold is now brieflydescribed in accordance with certain aspects of the invention.Additional details of the components forming the cell scaffold will bediscussed in more detail below, including various techniques forfabricating such cell scaffolds. In FIG. 1A, cell scaffold 10 is showncomprising nanoscale wires 15 and polymeric constructs 20. The nanoscalewires may be semiconductor nanowires (e.g., comprising silicon), and thepolymeric constructs can include photoresist polymers (such as SU-8),and/or biocompatible polymers (such as Matrigel™). Some of polymericconstructs 20 may contain conductive pathways 25 in electricalcommunication with some of the nanoscale wires 15, and the conductivepathways can extend externally of the surface of the cell scaffold, asis shown with conductive pathway 27. For example, some of the conductivepathways may be in electrical communication with external electricalsystem 30 such as a computer or a transmitter, e.g., such that physicaland/or electrical properties of the nanoscale wires can be determined,and/or such that electrical stimuli can be applied to the nanoscalewires. Thus, the conductive pathways and nanoscale wires may form partof an electrical circuit in some cases. In certain embodiments, theconductive pathways can be formed out of metal leads, and in some casesdissimilar metals (e.g., chromium and palladium) can be used, e.g., tocause the cell scaffold to adopt a 3-dimensional structure. Accordingly,the cell scaffold may be constructed and arranged to havecharacteristics suitable for growing cells or tissues, e.g., cells 35 inFIG. 1A. For example, the cell scaffold may have an open porosity of atleast about 50%, and/or an average pore size between about 100micrometers and about 1.5 mm.

In some cases, at least a portion of the cell scaffold is formed frombiodegradable materials, which can degrade over time. Thus, for example,referring now to FIG. 1B, cell scaffold 10 from FIG. 1A (containingcells 35 and being in electrical communication with external electricalsystem 30) may begin to degrade, eventually leaving behind nondegradablecomponents of the cell scaffold, e.g., nondegradable polymers 18,nanoscale wires 15, conductive pathways 25, and the like, as is shownschematically in FIG. 1B. However, the nondegradable components may notnecessarily be degraded, and in some cases these components can continueto function. For example, nanoscale wires 15 may be connected viaconductive pathways 25 in electrical communication with externalelectrical system 30 such as a computer or a transmitter, even after thecell scaffold has been partially or fully degraded. Such nanoscale wirescan continue to be used, e.g., to determine properties of cells ortissue, to apply electrical stimuli to cells or tissue, etc., even afterpartial or complete degradation of the cell scaffold.

In one set of embodiments, such cell scaffolds are formed by depositingpolymers and/or metals on a sacrificial material, which is then removed,as is illustrated in FIG. 1C. Briefly, on substrate 70, various polymers80, metals 85, nanoscale wires 88, and/or other components are assembledinto structure 90, at least a portion of which is present on sacrificialmaterial 75 on substrate 70. Some or all of sacrificial material 75 canbe subsequently removed to release structure 90. In some embodiments,structure 90 may then be formed into 3-dimensional structure 95 for thecell scaffold, for example, spontaneously, by folding or rolling thestructure, by applying stresses to the 2-dimensional structure thatcause it to adopt a 3-dimensional configuration, or the like. Forinstance, by pre-stressing certain components within the structure, uponremoval of the sacrificial material, the structure can spontaneouslyform a 3-dimensional structure. In some embodiments, various materialsare added to the structure (before or after removal of the sacrificialmaterial), for example, to help stabilize the structure, to addadditional agents to enhance its biocompatibility (e.g., growthhormones, extracellular matrix protein, Matrigel™, etc.), or the like.

The above discussion is just a brief summary of some embodiments of thepresent invention. However, it should be understood that otherembodiments are also possible in addition to the ones described above,involving various types of materials, techniques for forming cellscaffolds, applications, and the like, which will now be discussed ingreater detail.

In general, cell scaffolds are structures that cells can attach to andgrow on, e.g., to form biological tissues and other biologicalstructures. The cell scaffold may comprise biocompatible and/orbiodegradable materials in some aspects of the invention, and may alsocontain growth factors such as growth hormones, extracellular matrixproteins, specific metabolites or nutrients, or the like. The cellscaffold typically is porous, e.g., to facilitate cell seeding therein,and/or diffusion into and out of the cell scaffold, e.g., of nutrients,waste products, etc.

The cell scaffold can also comprise one or more nanoscale wires.Non-limiting examples of suitable nanoscale wires include carbonnanotubes, nanorods, nanowires, organic and inorganic conductive andsemiconducting polymers, metal nanoscale wires, semiconductor nanoscalewires (for example, formed from silicon), and the like. If carbonnanotubes are used, they may be single-walled and/or multi-walled, andmay be metallic and/or semiconducting in nature. Other conductive orsemiconducting elements that may not be nanoscale wires, but are ofvarious small nanoscopic-scale dimension, also can be used within thecell scaffold.

In general, a “nanoscale wire” (also known herein as a “nanoscopic-scalewire” or “nanoscopic wire”) generally is a wire or other nanoscaleobject, that at any point along its length, has at least onecross-sectional dimension and, in some embodiments, two orthogonalcross-sectional dimensions (e.g., a diameter) of less than 1 micrometer,less than about 500 nm, less than about 200 nm, less than about 150 nm,less than about 100 nm, less than about 70, less than about 50 nm, lessthan about 20 nm, less than about 10 nm, less than about 5 nm, thanabout 2 nm, or less than about 1 nm. In some embodiments, the nanoscalewire is generally cylindrical. In other embodiments, however, othershapes are possible; for example, the nanoscale wire can be faceted,i.e., the nanoscale wire may have a polygonal cross-section. Thecross-section of a nanoscale wire can be of any arbitrary shape,including, but not limited to, circular, square, rectangular, annular,polygonal, or elliptical, and may be a regular or an irregular shape.The nanoscale wire can also be solid or hollow.

In some cases, the nanoscale wire has one dimension that issubstantially longer than the other dimensions of the nanoscale wire.For example, the nanoscale wire may have a longest dimension that is atleast about 1 micrometer, at least about 3 micrometers, at least about 5micrometers, or at least about 10 micrometers or about 20 micrometers inlength, and/or the nanoscale wire may have an aspect ratio (longestdimension to shortest orthogonal dimension) of greater than about 2:1,greater than about 3:1, greater than about 4:1, greater than about 5:1,greater than about 10:1, greater than about 25:1, greater than about50:1, greater than about 75:1, greater than about 100:1, greater thanabout 150:1, greater than about 250:1, greater than about 500:1, greaterthan about 750:1, or greater than about 1000:1 or more in some cases.

In some embodiments, a nanoscale wire are substantially uniform, or havea variation in average diameter of the nanoscale wire of less than about30%, less than about 25%, less than about 20%, less than about 15%, lessthan about 10%, or less than about 5%. For example, the nanoscale wiresmay be grown from substantially uniform nanoclusters or particles, e.g.,colloid particles. See, e.g., U.S. Pat. No. 7,301,199, issued Nov. 27,2007, entitled “Nanoscale Wires and Related Devices,” by Lieber, et al.,incorporated herein by reference in its entirety. In some cases, thenanoscale wire may be one of a population of nanoscale wires having anaverage variation in diameter, of the population of nanowires, of lessthan about 30%, less than about 25%, less than about 20%, less thanabout 15%, less than about 10%, or less than about 5%.

In some embodiments, a nanoscale wire has a conductivity of or ofsimilar magnitude to any semiconductor or any metal. The nanoscale wirecan be formed of suitable materials, e.g., semiconductors, metals, etc.,as well as any suitable combinations thereof. In some cases, thenanoscale wire will have the ability to pass electrical charge, forexample, being electrically conductive. For example, the nanoscale wiremay have a relatively low resistivity, e.g., less than about 10⁻³ Ohm m,less than about 10⁻⁴ Ohm m, less than about 10⁻⁶ Ohm m, or less thanabout 10⁻⁷ Ohm m. The nanoscale wire can, in some embodiments, have aconductance of at least about 1 microsiemens, at least about 3microsiemens, at least about 10 microsiemens, at least about 30microsiemens, or at least about 100 microsiemens.

The nanoscale wire can be solid or hollow, in various embodiments. Asused herein, a “nanotube” is a nanoscale wire that is hollow, or thathas a hollowed-out core, including those nanotubes known to those ofordinary skill in the art. As another example, a nanotube may be createdby creating a core/shell nanowire, then etching away at least a portionof the core to leave behind a hollow shell. Accordingly, in one set ofembodiments, the nanoscale wire is a non-carbon nanotube. In contrast, a“nanowire” is a nanoscale wire that is typically solid (i.e., nothollow). Thus, in one set of embodiments, the nanoscale wire may be asemiconductor nanowire, such as a silicon nanowire.

For example, in one embodiment, a nanoscale wire may comprise or consistessentially of a metal. Non-limiting examples of potentially suitablemetals include aluminum, gold, silver, copper, molybdenum, tantalum,titanium, nickel, tungsten, chromium, or palladium. In another set ofembodiments, a nanoscale wire comprises or consists essentially of asemiconductor. Typically, a semiconductor is an element havingsemiconductive or semi-metallic properties (i.e., between metallic andnon-metallic properties). An example of a semiconductor is silicon.Other non-limiting examples include elemental semiconductors, such asgallium, germanium, diamond (carbon), tin, selenium, tellurium, boron,or phosphorous. In other embodiments, more than one element may bepresent in the nanoscale wire as the semiconductor, for example, galliumarsenide, gallium nitride, indium phosphide, cadmium selenide, etc.Still other examples include a Group II-VI material (which includes atleast one member from Group II of the Periodic Table and at least onemember from Group VI, for example, ZnS, ZnSe, ZnSSe, ZnCdS, CdS, orCdSe), or a Group III-V material (which includes at least one memberfrom Group III and at least one member from Group V, for example GaAs,GaP, GaAsP, InAs, InP, AlGaAs, or InAsP).

In certain embodiments, the semiconductor can be undoped or doped (e.g.,p-type or n-type). For example, in one set of embodiments, a nanoscalewire may be a p-type semiconductor nanoscale wire or an n-typesemiconductor nanoscale wire, and can be used as a component of atransistor such as a field effect transistor (“FET”). For instance, thenanoscale wire may act as the “gate” of a source-gate-drain arrangementof a FET, while metal leads or other conductive pathways (as discussedherein) are used as the source and drain electrodes.

In some embodiments, a dopant or a semiconductor may include mixtures ofGroup IV elements, for example, a mixture of silicon and carbon, or amixture of silicon and germanium. In other embodiments, the dopant orthe semiconductor may include a mixture of a Group III and a Group Velement, for example, BN, BP, BAs, AN, AlP, AlAs, AlSb, GaN, GaP, GaAs,GaSb, InN, InP, InAs, or InSb. Mixtures of these may also be used, forexample, a mixture of BN/BP/BAs, or BN/AlP. In other embodiments, thedopants may include alloys of Group III and Group V elements. Forexample, the alloys may include a mixture of AlGaN, GaPAs, InPAs, GaInN,AlGaInN, GaInAsP, or the like. In other embodiments, the dopants mayalso include a mixture of Group II and Group VI semiconductors. Forexample, the semiconductor may include ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe,CdTe, HgS, HgSe, HgTe, BeS, BeSe, BeTe, MgS, MgSe, or the like. Alloysor mixtures of these dopants are also be possible, for example,(ZnCd)Se, or Zn(SSe), or the like. Additionally, alloys of differentgroups of semiconductors may also be possible, for example, acombination of a Group II-Group VI and a Group III-Group Vsemiconductor, for example, (GaAs)_(x)(ZnS)_(1-x). Other examples ofdopants may include combinations of Group IV and Group VI elements, suchas GeS, GeSe, GeTe, SnS, SnSe, SnTe, PbO, PbS, PbSe, or PbTe. Othersemiconductor mixtures may include a combination of a Group I and aGroup VII, such as CuF, CuCl, CuBr, CuI, AgF, AgCl, AgBr, AgI, or thelike. Other dopant compounds may include different mixtures of theseelements, such as BeSiN₂, CaCN₂, ZnGeP₂, CdSnAs₂, ZnSnSb₂, CuGeP₃,CuSi₂P₃, Si₃N₄, Ge₃N₄, Al₂O₃, (Al, Ga, In)₂(S, Se, Te)₃, Al₂CO, (Cu,Ag)(Al, Ga, In, Tl, Fe)(S, Se, Te)₂ and the like.

The doping of the semiconductor to produce a p-type or n-typesemiconductor may be achieved via bulk-doping in certain embodiments,although in other embodiments, other doping techniques (such as ionimplantation) can be used. Many such doping techniques that can be usedwill be familiar to those of ordinary skill in the art, including bothbulk doping and surface doping techniques. A bulk-doped article (e.g. anarticle, or a section or region of an article) is an article for which adopant is incorporated substantially throughout the crystalline latticeof the article, as opposed to an article in which a dopant is onlyincorporated in particular regions of the crystal lattice at the atomicscale, for example, only on the surface or exterior. For example, somearticles are typically doped after the base material is grown, and thusthe dopant only extends a finite distance from the surface or exteriorinto the interior of the crystalline lattice. It should be understoodthat “bulk-doped” does not define or reflect a concentration or amountof doping in a semiconductor, nor does it necessarily indicate that thedoping is uniform. “Heavily doped” and “lightly doped” are terms themeanings of which are clearly understood by those of ordinary skill inthe art. In some embodiments, one or more regions comprise a singlemonolayer of atoms (“delta-doping”). In certain cases, the region may beless than a single monolayer thick (for example, if some of the atomswithin the monolayer are absent). As a specific example, the regions maybe arranged in a layered structure within the nanoscale wire, and one ormore of the regions can be delta-doped or partially delta-doped.

Accordingly, in one set of embodiments, the nanoscale wires may includea heterojunction, e.g., of two regions with dissimilar materials orelements, and/or the same materials or elements but at different ratiosor concentrations. The regions of the nanoscale wire may be distinctfrom each other with minimal cross-contamination, or the composition ofthe nanoscale wire can vary gradually from one region to the next. Theregions may be both longitudinally arranged relative to each other, orradially arranged (e.g., as in a core/shell arrangement) on thenanoscale wire. Each region may be of any size or shape within the wire.The junctions may be, for example, a p/n junction, a p/p junction, ann/n junction, a p/i junction (where i refers to an intrinsicsemiconductor), an n/i junction, an i/i junction, or the like. Thejunction can also be a Schottky junction in some embodiments. Thejunction may also be, for example, a semiconductor/semiconductorjunction, a semiconductor/metal junction, a semiconductor/insulatorjunction, a metal/metal junction, a metal/insulator junction, aninsulator/insulator junction, or the like. The junction may also be ajunction of two materials, a doped semiconductor to a doped or anundoped semiconductor, or a junction between regions having differentdopant concentrations. The junction can also be a defected region to aperfect single crystal, an amorphous region to a crystal, a crystal toanother crystal, an amorphous region to another amorphous region, adefected region to another defected region, an amorphous region to adefected region, or the like. More than two regions may be present, andthese regions may have unique compositions or may comprise the samecompositions. As one example, a wire can have a first region having afirst composition, a second region having a second composition, and athird region having a third composition or the same composition as thefirst composition. Non-limiting examples of nanoscale wires comprisingheterojunctions (including core/shell heterojunctions, longitudinalheterojunctions, etc., as well as combinations thereof) are discussed inU.S. Pat. No. 7,301,199, issued Nov. 27, 2007, entitled “Nanoscale Wiresand Related Devices,” by Lieber, et al., incorporated herein byreference in its entirety.

In some embodiments, a nanoscale wire is a bent or a kinked nanoscalewire. A kink is typically a relatively sharp transition or turningbetween a first substantially straight portion of a wire and a secondsubstantially straight portion of a wire. For example, a nanoscale wiremay have 1, 2, 3, 4, or 5 or more kinks. In some cases, the nanoscalewire is formed from a single crystal and/or comprises or consistsessentially of a single crystallographic orientation, for example, a<110> crystallographic orientation, a <112> crystallographicorientation, or a <1120> crystallographic orientation. It should benoted that the kinked region need not have the same crystallographicorientation as the rest of the semiconductor nanoscale wire. In someembodiments, a kink in the semiconductor nanoscale wire may be at anangle of about 120° or a multiple thereof. The kinks can beintentionally positioned along the nanoscale wire in some cases. Forexample, a nanoscale wire may be grown from a catalyst particle byexposing the catalyst particle to various gaseous reactants to cause theformation of one or more kinks within the nanoscale wire. Non-limitingexamples of kinked nanoscale wires, and suitable techniques for makingsuch wires, are disclosed in International Patent Application No.PCT/US2010/050199, filed Sep. 24, 2010, entitled “Bent Nanowires andRelated Probing of Species,” by Tian, et al., published as WO2011/038228 on Mar. 31, 2011, incorporated herein by reference in itsentirety.

In one set of embodiments, the nanoscale wire is formed from a singlecrystal, for example, a single crystal nanoscale wire comprising asemiconductor. A single crystal item may be formed via covalent bonding,ionic bonding, or the like, and/or combinations thereof. While such asingle crystal item may include defects in the crystal in some cases,the single crystal item is distinguished from an item that includes oneor more crystals, not ionically or covalently bonded, but merely inclose proximity to one another.

In some embodiments, the nanoscale wires used herein are individual orfree-standing nanoscale wires. For example, an “individual” or a“free-standing” nanoscale wire may, at some point in its life, not beattached to another article, for example, with another nanoscale wire,or the free-standing nanoscale wire may be in solution. This is incontrast to nanoscale features etched onto the surface of a substrate,e.g., a silicon wafer, in which the nanoscale features are never removedfrom the surface of the substrate as a free-standing article. This isalso in contrast to conductive portions of articles which differ fromsurrounding material only by having been altered chemically orphysically, in situ, i.e., where a portion of a uniform article is madedifferent from its surroundings by selective doping, etching, etc. An“individual” or a “free-standing” nanoscale wire is one that can be (butneed not be) removed from the location where it is made, as anindividual article, and transported to a different location and combinedwith different components to make a functional device such as thosedescribed herein and those that would be contemplated by those ofordinary skill in the art upon reading this disclosure.

In various embodiments, more than one nanoscale wire may be presentwithin the cell scaffold. The nanoscale wires may each independently bethe same or different. For example, the cell scaffold can comprise atleast 5 nanoscale wires, at least about 10 nanoscale wires, at leastabout 30 nanoscale wires, at least about 50 nanoscale wires, at leastabout 100 nanoscale wires, at least about 300 nanoscale wires, at leastabout 1000 nanoscale wires, etc. The nanoscale wires may be distributeduniformly or non-uniformly throughout the cell scaffold. In some cases,the nanoscale wires may be distributed at an average density of at leastabout 10 nanoscale wires/mm³, at least about 30 nanoscale wires/mm³, atleast about 50 nanoscale wires/mm³, at least about 75 nanoscalewires/mm³, or at least about 100 nanoscale wires/mm³. In certainembodiments, the nanoscale wires are distributed within the cellscaffold such that the average separation between a nanoscale wire andits nearest neighboring nanoscale wire is less than about 2 mm, lessthan about 1 mm, less than about 500 micrometers, less than about 300micrometers, less than about 100 micrometers, less than about 50micrometers, less than about 30 micrometers, or less than about 10micrometers.

Within the cell scaffold, some or all of the nanoscale wires may beindividually electronically addressable. For instance, in some cases, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, or substantially all of thenanoscale wires within the cell scaffold may be individuallyelectronically addressable. In some embodiments, an electrical propertyof a nanoscale wire can be individually determinable (e.g., beingpartially or fully resolvable without also including the electricalproperties of other nanoscale wires), and/or such that the electricalproperty of a nanoscale wire may be individually controlled (e.g., byapplying a desired voltage or current to the nanoscale wire, forinstance, without simultaneously applying the voltage or current toother nanoscale wires). In other embodiments, however, at least some ofthe nanoscale wires can be controlled within the same electronic circuit(e.g., by incorporating the nanoscale wires in series and/or inparallel), such that the nanoscale wires can still be electronicallycontrolled and/or determined.

The nanoscale wire, in some embodiments, may be responsive to a propertyexternal of the nanoscale wire, e.g., a chemical property, an electricalproperty, a physical property, etc. Such determination may bequalitative and/or quantitative. For example, in one set of embodiments,the nanoscale wire may be responsive to voltage. For instance, thenanoscale wire may exhibits a voltage sensitivity of at least about 5microsiemens/V; by determining the conductivity of a nanoscale wire, thevoltage surrounding the nanoscale wire may thus be determined. In otherembodiments, the voltage sensitivity can be at least about 10microsiemens/V, at least about 30 microsiemens/V, at least about 50microsiemens/V, or at least about 100 microsiemens/V. Other examples ofelectrical properties that can be determined include resistance,resistivity, conductance, conductivity, impendence, or the like.

As another example, a nanoscale wire may be responsive to a chemicalproperty of the environment surrounding the nanoscale wire. For example,an electrical property of the nanoscale wire can be affected by achemical environment surrounding the nanoscale wire, and the electricalproperty can be thereby determined to determine the chemical environmentsurrounding the nanoscale wire. As a specific non-limiting example, thenanoscale wires may be sensitive to pH or hydrogen ions. Furthernon-limiting examples of such nanoscale wires are discussed in U.S. Pat.No. 7,129,554, filed Oct. 31, 2006, entitled “Nanosensors,” by Lieber,et al., incorporated herein by reference in its entirety.

As an example, the nano scale wire may have the ability to bind to ananalyte indicative of a chemical property of the environment surroundingthe nanoscale wire (e.g., hydrogen ions for pH, or concentration for ananalyte of interest), and/or the nanoscale wire may be partially orfully functionalized, i.e. comprising surface functional moieties, towhich an analyte is able to bind, thereby causing a determinableproperty change to the nanoscale wire, e.g., a change to the resistivityor impedance of the nanoscale wire. The binding of the analyte can bespecific or non-specific. Functional moieties may include simple groups,selected from the groups including, but not limited to, —OH, —CHO,—COOH, —SO₃H, —CN, —NH₂, —SH, —COSH, —COOR, halide; biomolecularentities including, but not limited to, amino acids, proteins, sugars,DNA, antibodies, antigens, and enzymes; grafted polymer chains withchain length less than the diameter of the nanowire core, selected froma group of polymers including, but not limited to, polyamide, polyester,polyimide, polyacrylic; a shell of material comprising, for example,metals, semiconductors, and insulators, which may be a metallic element,an oxide, an sulfide, a nitride, a selenide, a polymer and a polymergel.

In some embodiments, a reaction entity may be bound to a surface of thenanoscale wire, and/or positioned in relation to the nanoscale wire suchthat the analyte can be determined by determining a change in a propertyof the nanoscale wire. The “determination” may be quantitative and/orqualitative, depending on the application. The term “reaction entity”refers to any entity that can interact with an analyte in such a mannerto cause a detectable change in a property (such as an electricalproperty) of a nanoscale wire. The reaction entity may enhance theinteraction between the nanowire and the analyte, or generate a newchemical species that has a higher affinity to the nanowire, or toenrich the analyte around the nanowire. The reaction entity can comprisea binding partner to which the analyte binds. The reaction entity, whena binding partner, can comprise a specific binding partner of theanalyte. For example, the reaction entity may be a nucleic acid, anantibody, a sugar, a carbohydrate or a protein. Alternatively, thereaction entity may be a polymer, catalyst, or a quantum dot. A reactionentity that is a catalyst can catalyze a reaction involving the analyte,resulting in a product that causes a detectable change in the nanowire,e.g. via binding to an auxiliary binding partner of the productelectrically coupled to the nanowire. Another exemplary reaction entityis a reactant that reacts with the analyte, producing a product that cancause a detectable change in the nanowire. The reaction entity cancomprise a shell on the nanowire, e.g. a shell of a polymer thatrecognizes molecules in, e.g., a gaseous sample, causing a change inconductivity of the polymer which, in turn, causes a detectable changein the nanowire.

The term “binding partner” refers to a molecule that can undergo bindingwith a particular analyte, or “binding partner” thereof, and includesspecific, semi-specific, and non-specific binding partners as known tothose of ordinary skill in the art. The term “specifically binds,” whenreferring to a binding partner (e.g., protein, nucleic acid, antibody,etc.), refers to a reaction that is determinative of the presence and/oridentity of one or other member of the binding pair in a mixture ofheterogeneous molecules (e.g., proteins and other biologics). Thus, forexample, in the case of a receptor/ligand binding pair the ligand wouldspecifically and/or preferentially select its receptor from a complexmixture of molecules, or vice versa. An enzyme would specifically bindto its substrate, a nucleic acid would specifically bind to itscomplement, an antibody would specifically bind to its antigen. Otherexamples include, nucleic acids that specifically bind (hybridize) totheir complement, antibodies specifically bind to their antigen, and thelike. The binding may be by one or more of a variety of mechanismsincluding, but not limited to ionic interactions, and/or covalentinteractions, and/or hydrophobic interactions, and/or van der Waalsinteractions, etc.

Some or all of the nanoscale wires may be in electrical communicationwith a surface of the cell scaffold via one or more conductive pathways.In some embodiments, conductive pathways can be used to determine aproperty of a nanoscale wire (for example, an electrical property or achemical property as is discussed herein), and/or the conductive pathwaymay be used to direct an electrical signal to the nanoscale wire, e.g.,to electrically stimulate cells proximate the nanoscale wire. Theconductive pathways can form an electrical circuit that is internallycontained within the cell scaffold, and/or that extends externally ofthe cell scaffold, e.g., such that the electrical circuit is inelectrical communication with an external electrical system, such as acomputer or a transmitter (for instance, a radio transmitter, a wirelesstransmitter, an Internet connection, etc.). Any suitable pathwayconductive pathway may be used, for example, pathways comprising metals,semiconductors, conductive polymers, or the like.

In some embodiments, more than one conductive pathway may be used withina cell scaffold. For example, multiple conductive pathways can be usedsuch that some or all of the nanoscale wires may be individuallyelectronically addressable within the cell scaffold. However, in otherembodiments, more than one nanoscale wire may be addressable by aparticular conductive pathway. In addition, in some cases, otherelectronic components may also be present within the cell scaffold,e.g., as part of a conductive pathway or otherwise forming part of anelectrical circuit. Examples include, but are not limited to,transistors such as field effect transistors, resistors, capacitors,inductors, diodes, integrated circuits, etc. In some cases, some ofthese may also comprise nanoscale wires.

In addition, in some cases, the conductive pathway and/or electroniccomponents can be at least partially surrounded by or contained withinone or more polymeric constructs used to form the cell scaffold. Forexample, a conductive pathway, such as a metal lead, may be “sandwiched”between two polymers (which can be the same or different from eachother) that form a polymeric construct of the cell scaffold.Accordingly, in some embodiments, the conductive pathway may berelatively narrow. For example, the conductive pathway may have asmallest dimension or a largest cross-sectional dimension of less thanabout 5 micrometers, less than about 4 micrometers, less than about 3micrometers, less than about 2 micrometers, less than about 1micrometer, less than about 700 nm, less than about 600 nm, less thanabout 500 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm, less than about 80 nm, less than about 50 nm, less thanabout 30 nm, less than about 10 nm, less than about 5 nm, less thanabout 2 nm, etc. The conductive pathway may have any suitablecross-sectional shape, e.g., circular, square, rectangular, polygonal,elliptical, regular, irregular, etc. As is discussed in detail below,such conductive pathways may be achieved using lithographic or othertechniques.

A given conductive pathway within a cell scaffold may be in electricalcommunication with any number of nanoscale wires within a cell scaffold,depending on the embodiment. For example, a conductive pathway can be inelectrical communication with one, two, three, or more nanoscale wires,and if more than one nanoscale wire is used within a given conductivepathway, the nanoscale wires may each independently be the same ordifferent. Thus, for example, an electrical property of the nanoscalewire may be determined via the conductive pathway, and/or a signal canbe propagated via the conductive pathway to the nanoscale wire. Inaddition, as previously discussed, some or all of the nanoscale wiresmay be in electrical communication with a surface of the cell scaffoldvia one or more conductive pathways. For example, in some cases, atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90% of the nanoscale wires within thecell scaffold may be in electrical communication with one or moreconductive pathways, or otherwise form portions of one or moreelectrical circuits extending externally of the cell scaffold. In somecases, however, not all of the nanoscale wires within a cell scaffoldmay be in electrical communication with one or more conductive pathways,e.g., by design, or because of inefficiencies within the fabricationprocess, etc.

In some embodiments, one or more metal leads can be used within aconductive pathway to a nanoscale wire. The metal lead may directlyphysically contact the nanoscale wire and/or there may be othermaterials between the metal lead and the nanoscale wire that allowelectrical communication to occur. Metal leads are useful due to theirhigh conductance, e.g., such that changes within electrical propertiesobtained from the conductive pathway can be related to changes inproperties of the nanoscale wire, rather than changes in properties ofthe conductive pathway. However, it is not a requirement that only metalleads be used, and in other embodiments, other types of conductivepathways may also be used, in addition or instead of metal leads.

A wide variety of metal leads can be used, in various embodiments of theinvention. As non-limiting examples, the metals used within a metal leadmay include aluminum, gold, silver, copper, molybdenum, tantalum,titanium, nickel, tungsten, chromium, palladium, as well as anycombinations of these and/or other metals. In some cases, the metal canbe chosen to be one that is readily introduced into the cell scaffold,e.g., using techniques compatible with lithographic techniques. Forexample, in one set of embodiments, lithographic techniques such ase-beam lithography, photolithography, X-ray lithography, extremeultraviolet lithography, ion projection lithography, etc. may be used tolayer or deposit one or more metals on a substrate. Additionalprocessing steps can also be used to define or register the metal leadsin some cases. Thus, for example, the thickness of a metal layer may beless than about 5 micrometers, less than about 4 micrometers, less thanabout 3 micrometers, less than about 2 micrometers, less than about 1micrometer, less than about 700 nm, less than about 600 nm, less thanabout 500 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm, less than about 80 nm, less than about 50 nm, less thanabout 30 nm, less than about 10 nm, less than about 5 nm, less thanabout 2 nm, etc. The thickness of the layer may also be at least about10 nm, at least about 20 nm, at least about 40 nm, at least about 60 nm,at least about 80 nm, or at least about 100 nm. For example, thethickness of a layer may be between about 40 nm and about 100 nm,between about 50 nm and about 80 nm.

In some embodiments, more than one metal can be used within a metallead. For example, two, three, or more metals may be used within a metallead. The metals may be deposited in different regions or alloyedtogether, or in some cases, the metals may be layered on top of eachother, e.g., layered on top of each other using various lithographictechniques. For example, a second metal may be deposited on a firstmetal, and in some cases, a third metal may be deposited on the secondmetal, etc. Additional layers of metal (e.g., fourth, fifth, sixth,etc.) may also be used in some embodiments. The metals can all bedifferent, or in some cases, some of the metals (e.g., the first andthird metals) may be the same. Each layer may independently be of anysuitable thickness or dimension, e.g., of the dimensions describedabove, and the thicknesses of the various layers can independently bethe same or different.

If dissimilar metals are layered on top of each other, they may belayered in some embodiments in a “stressed” configuration (although inother embodiments they may not necessarily be stressed). As a specificnon-limiting example, chromium and palladium can be layered together tocause stresses in the metal leads to occur, thereby causing warping orbending of the metal leads. The amount and type of stress may also becontrolled, e.g., by controlling the thicknesses of the layers. Forexample, relatively thinner layers can be used to increase the amount ofwarping that occurs.

Without wishing to be bound by any theory, it is believed that layeringmetals having a difference in stress (e.g., film stress) with respect toeach other may, in some cases, cause stresses within the metal, whichcan cause bending or warping as the metals seek to relieve the stresses.In some embodiments, such mismatches are undesirable because they couldcause warping of the metal leads and thus, the cell scaffold. However,in other embodiments, such mismatches may be desired, e.g., so that thecell scaffold can be intentionally deformed to form a 3-dimensionalstructure, as discussed below. In addition, in certain embodiments, thedeposition of mismatched metals within a lead may occur at specificlocations within the cell scaffold, e.g., to cause specific warpings tooccur, which can be used to cause the cell scaffold to be deformed intoa particular shape or configuration. For example, a “line” of suchmismatches can be used to cause an intentional bending or folding alongthe line of the cell scaffold.

In one set of embodiments, the cell scaffold may also contain one ormore polymeric constructs. The polymeric constructs typically compriseone or more polymers, e.g., photoresists, biocompatible polymers,biodegradable polymers, etc., and optionally may contain othermaterials, for example, metal leads or other conductive pathwaymaterials. The polymeric constructs may be separately formed thenassembled into a cell scaffold, and/or the polymeric constructs may beintegrally formed as part of the cell scaffold, for example, by formingor manipulating (e.g. folding, rolling, etc.) the polymeric constructsinto a 3-dimensional structure that defines the cell scaffold.

In one set of embodiments, some or all of the polymeric constructs havethe form of fibers or ribbons. For example, the polymeric constructs mayhave one dimension that is substantially longer than the otherdimensions of the polymeric construct. The fibers can in some cases bejoined together to form a “network” or “mesh” of fibers that define thecell scaffold. For example, referring to FIG. 4A, Panel II, a cellscaffold may contain a plurality of fibers that are orthogonallyarranged to form a regular network of polymeric constructs. However, thepolymeric constructs need not be regularly arranged, as is shown in FIG.4A, Panel I, with a more irregular arrangement of polymer constructs. Inaddition, it should be noted that although FIG. 4A shows only polymerconstructs having the form of fibers, this is by way of example only,and in other embodiments, other shapes of polymeric constructs can beused. In general, any shape or dimension of polymeric construct thatallows for cell growth may be used.

Thus, for example, in one set of embodiments, some or all of thepolymeric constructs have a smallest dimension or a largestcross-sectional dimension of less than about 5 micrometers, less thanabout 4 micrometers, less than about 3 micrometers, less than about 2micrometers, less than about 1 micrometer, less than about 700 nm, lessthan about 600 nm, less than about 500 nm, less than about 300 nm, lessthan about 200 nm, less than about 100 nm, less than about 80 nm, lessthan about 50 nm, less than about 30 nm, less than about 10 nm, lessthan about 5 nm, less than about 2 nm, etc. A polymeric construct mayalso have any suitable cross-sectional shape, e.g., circular, square,rectangular, polygonal, elliptical, regular, irregular, etc. Examples ofmethods of forming polymeric constructs, e.g., by lithographic or othertechniques, are discussed below.

In one set of embodiment, the polymeric constructs can be arranged suchthat the cell scaffold has dimensions that facilitate cell seedingtherein, and/or diffusion into and out of the cell scaffold, e.g., ofnutrients, waste products, etc. For example, in some cases, thepolymeric constructs may be constructed and arranged within the cellscaffold such that the cell scaffold has an open porosity of at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97, atleast about 99%, at least about 99.5%, or at least about 99.8%. The“open porosity” is generally described as the volume of empty spacewithin the cell scaffold divided by the overall volume defined by thecell scaffold, and can be thought of as being equivalent to void volume.Typically, the open porosity includes the volume within the cellscaffold to which cells can access. In some cases, the cell scaffolddoes not contain significant amounts of internal volume to which thecells are incapable of addressing, e.g., due to lack of access and/orpore access being too small.

In some cases, a “two-dimensional open porosity” may also be defined,e.g., of a cell scaffold that is subsequently formed or manipulated intoa 3-dimensional structure. The two-dimensional open porosities of a cellscaffold can be defined as the void area within the two-dimensionalconfiguration of the cell scaffold (e.g., where no material is present)divided by the overall area of cell scaffold, and can be determinedbefore or after the cell scaffold has been formed into a 3-dimensionalstructure. Depending on the application, a cell scaffold may have atwo-dimensional open porosity of at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 97, at least about 99%, at leastabout 99.5%, or at least about 99.8%, etc.

Another method of generally determining the two-dimensional porosity ofthe cell scaffold is by determining the areal mass density, i.e., themass of the cell scaffold divided by the area of one face of the cellscaffold (including holes or voids present therein). Thus, for example,in another set of embodiments, the cell scaffold may have an areal massdensity of less than about 100 micrograms/cm², less than about 80micrograms/cm², less than about 60 micrograms/cm², less than about 50micrograms/cm², less than about 40 micrograms/cm², less than about 30micrograms/cm², or less than about 20 micrograms/cm².

The porosity of a cell scaffold can be defined by one or more pores.Pores that are too small can hinder or restrict cell access. Thus, inone set of embodiments, the cell scaffold may have an average pore sizeof at least about 100 micrometers, at least about 200 micrometers, atleast about 300 micrometers, at least about 400 micrometers, at leastabout 500 micrometers, at least about 600 micrometers, at least about700 micrometers, at least about 800 micrometers, at least about 900micrometers, or at least about 1 mm. However, in other embodiments,pores that are too big may prevent cells from being able tosatisfactorily use or even access the pore volume. Thus, in some cases,the cell scaffold may have an average pore size of no more than about1.5 mm, no more than about 1.4 mm, no more than about 1.3 mm, no morethan about 1.2 mm, no more than about 1.1 mm, no more than about 1 mm,no more than about 900 micrometers, no more than about 800 micrometers,no more than about 700 micrometers, no more than about 600 micrometers,or no more than about 500 micrometers. Combinations of these are alsopossible, e.g., in one embodiment, the average pore size is at leastabout 100 micrometers and no more than about 1.5 mm. In addition, largeror smaller pores than these can also be used in a cell scaffold incertain cases. Pore sizes may be determined using any suitabletechnique, e.g., through visual inspection, BET measurements, or thelike.

In various embodiments, one or more of the polymers forming a polymericconstruct may be a photoresist. While not commonly used in cellscaffolds, photoresists are typically used in lithographic techniques,which can be used as discussed herein to form the polymeric construct.For example, the photoresist may be chosen for its ability to react tolight to become substantially insoluble (or substantially soluble, insome cases) to a photoresist developer. For instance, photoresists thatcan be used within a polymeric construct include, but are not limitedto, SU-8, S1805, LOR 3A, poly(methyl methacrylate), poly(methylglutarimide), phenol formaldehyde resin (diazonaphthoquinone/novolac),diazonaphthoquinone (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley1400-17, Shipley 1400-27, Shipley 1400-37, or the like. These and manyother photoresists are available commercially.

A polymeric construct may also contain one or more polymers that arebiocompatible and/or biodegradable, in certain embodiments. A polymercan be biocompatible, biodegradable, or both biocompatible andbiodegradable, and in some cases, the degree of biodegradation orbiocompatibility depends on the physiological environment to which thepolymer is exposed to.

Typically, a biocompatible material is one that does not illicit animmune response, or elicits a relatively low immune response, e.g., onethat does not impair the cell scaffold or the cells therein fromcontinuing to function for its intended use. In some embodiments, thebiocompatible material is able to perform its desired function withouteliciting any undesirable local or systemic effects in the subject. Insome cases, the material can be incorporated into tissues within thesubject, e.g., without eliciting any undesirable local or systemiceffects, or such that any biological response by the subject does notsubstantially affect the ability of the material from continuing tofunction for its intended use. For example, in a cell scaffold, the cellscaffold may be able to support appropriate cellular or tissue activitywhen implanted within a subject, e.g., including the facilitation ofmolecular and/or mechanical signaling systems, without substantiallyeliciting undesirable effects in those cells, or undesirable local orsystemic responses, or without eliciting a response that causes the cellscaffold to cease functioning for its intended use. Examples oftechniques for determining biocompatibility include, but are not limitedto, the ISO 10993 series of for evaluating the biocompatibility ofmedical devices. As another example, a biocompatible material may beimplanted in a subject for an extended period of time, e.g., at leastabout a month, at least about 6 months, or at least about a year, andthe integrity of the material, or the immune response to the material,may be determined. For example, a suitably biocompatible material may beone in which the immune response is minimal, e.g., one that does notsubstantially harm the health of the subject. One example of abiocompatible material is poly(methyl methacrylate). In someembodiments, a biocompatible material may be used to cover or shield anon-biocompatible material (or a poorly biocompatible material) from thecells or tissue, for example, by covering the material.

A biodegradable material typically degrades over time when exposed to abiological system, e.g., through oxidation, hydrolysis, enzymaticattack, phagocytosis, or the like. For example, a biodegradable materialcan degrade over time when exposed to water (e.g., hydrolysis) orenzymes. In some cases, a biodegradable material is one that exhibitsdegradation (e.g., loss of mass and/or structure) when exposed tophysiological conditions for at least about a month, at least about 6months, or at least about a year. For example, the biodegradablematerial may exhibit a loss of mass of at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, or at leastabout 90%. In certain cases, some or all of the degradation products maybe resorbed or metabolized, e.g., into cells or tissues. For example,certain biodegradable materials, during degradation, release substancesthat can be metabolized by cells or tissues. For instance, polylacticacid releases water and lactic acid during degradation.

Examples of such biocompatible and/or biodegradable polymers include,but are not limited to, poly(lactic-co-glycolic acid), polylactic acid,polyglycolic acid, poly(methyl methacrylate), poly(trimethylenecarbonate), collagen, fibrin, polysaccharidic materials such as chitosanor glycosaminoglycans, hyaluronic acid, polycaprolactone, and the like.

The polymers and other components forming the cell scaffold can also beused in some embodiments to provide a certain degree of flexibility tothe cell scaffold, which can be quantified as a bending stiffness perunit width of polymer construct. An example method for determining thebending stiffness is discussed below. In various embodiments, the cellscaffold may have a bending stiffness of less than about 5 nN m, lessthan about 4.5 nN m, less than about 4 nN m, less than about 3.5 nN m,less than about 3 nN m, less than about 2.5 nN m, less than about 2 nNm, less than about 1.5 nN m, or less than about 1 nN m.

In some embodiments of the invention, the cell scaffold may also containother materials in addition to the photoresists or biocompatible and/orbiodegradable polymers described above. Non-limiting examples includeother polymers, growth hormones, extracellular matrix protein, specificmetabolites or nutrients, or the like. For example, in one ofembodiments, one or more agents able to promote cell growth can be addedto the cell scaffold, e.g., hormones such as growth hormones,extracellular matrix protein, pharmaceutical agents, vitamins, or thelike. Many such growth hormones are commercially available, and may bereadily selected by those of ordinary skill in the art based on thespecific type of cell or tissue used or desired. Similarly, non-limitingexamples of extracellular matrix proteins include gelatin, laminin,fibronectin, heparan sulfate, proteoglycans, entactin, hyaluronic acid,collagen, elastin, chondroitin sulfate, keratan sulfate, Matrigel™, orthe like. Many such extracellular matrix proteins are availablecommercially, and also can be readily identified by those of ordinaryskill in the art based on the specific type of cell or tissue used ordesired.

As another example, in one set of embodiments, additional scaffoldmaterials can be added to the cell scaffold, e.g., to control the sizeof pores within the cell scaffold, to promote cell adhesion or growthwithin the cell scaffold, to increase the structural stability of thecell scaffold, to control the flexibility of the cell scaffold, etc. Forinstance, in one set of embodiments, additional fibers or other suitablepolymers may be added to the cell scaffold, e.g., electrospun fibers canbe used as a secondary scaffold. The additional scaffold materials canbe formed from any of the materials described herein in reference tocell scaffolds, e.g., photoresists or biocompatible and/or biodegradablepolymers, or other polymers described herein. As another non-limitingexample, a glue such as a silicone elastomer glue can be used to controlthe shape of the cell scaffold.

In some cases, the cell scaffold can include a 2-dimensional structurethat is formed into a final 3-dimensional structure, e.g., by folding orrolling the structure. It should be understood that although the2-dimensional structure can be described as having an overall length,width, and height, the overall length and width of the structure mayeach be substantially greater than the overall height of the structure.The 2-dimensional structure may also be manipulated to have a differentshape that is 3-dimensional, e.g., having an overall length, width, andheight where the overall length and width of the structure are not eachsubstantially greater than the overall height of the structure. Forinstance, the structure may be manipulated to increase the overallheight of the material, relative to its overall length and/or width, forexample, by folding or rolling the structure. Thus, for example, arelatively planar sheet of material (having a length and width muchgreater than its thickness) may be rolled up into a “tube,” such thatthe tube has an overall length, width, and height of relativelycomparable dimensions).

Thus, for example, the 2-dimensional structure may comprise one or morenanoscale wires and one or more polymeric constructs formed into a2-dimensional structure or network that is subsequently formed into a3-dimensional structure. In some embodiments, the 2-dimesional structuremay be rolled or curled up to form the 3-dimesional structure, or the2-dimensional structure may be folded or creased one or more times toform the 3-dimesional structure. Such manipulations can be regular orirregular. In certain embodiments, as discussed herein, themanipulations are caused by pre-stressing the 2-dimensional structuresuch that it spontaneously forms the 3-dimensional structure, althoughin other embodiments, such manipulations can be performed separately,e.g., after formation of the 2-dimensional structure.

Cell scaffolds such as those described above can be used in a widevariety of applications, for example, for tissue engineering,prosthetics, pacemakers, implants, blood or other vessels, and the like.Accordingly, virtually any kind of cell that can be grown on a cellscaffold can be used, in various embodiments of the invention, e.g.,grown to form a tissue on the cell scaffold. In some cases, the cellsmay be ones that are electrically active, e.g., having electricalproperties which can be determined and/or controlled. Cells that areelectrically active include, but are not limited to, nerve cells orneurons, muscle cells, cardiac cells, or the like. However, in othercases, the cells do not necessarily have to be electrically active. Forexample, in one set of embodiments, chemical properties (such as pH) canbe determined using nanoscale wires, etc. that are contained within thecell scaffold, and the cells and/or tissues within the cell scaffoldaccordingly need not be electrically active (although they can be).

In one set of embodiments, a cell scaffold may not be present within abiological tissue (e.g., an implanted tissue), or may have been presentbut may have partially or completely degraded, e.g., such that it nolonger functions as a cell scaffold. Thus, for example, in oneembodiment, the present invention is directed to a biological tissuecomprising nanoscale wires such as semiconductor nanowires or any othernanoscale wire describe herein. In some cases, at least some of thenanoscale wires form a portion of an electrical circuit that extendsexternally of the tissue. The biological tissue may also compriseconductive pathways, such metal leads, within the biological tissue,e.g., connecting nanoscale wires or other electrical components. Inaddition, in some cases, some or all of the conductive pathways can alsobe connected to an external electrical system, such as a computer or atransmitter, e.g., a radio transmitter, a wireless transmitter, etc.Thus, in another set of embodiments, the present invention is generallydirected to a biological tissue comprising nanoscale wires and/orconductive pathways (e.g., forming an electrical network such as isdiscussed herein), not necessarily limited to a cell scaffold. Thetissue may be present in vitro or an in vivo, e.g., implanted into asubject, such as a human subject, the tissues may be autologous,homologous, or heterologous with the subject.

In some embodiments, cells or tissues can be interfaced with thenanoscale wires or other electrical components (within the cellscaffold, and/or after degradation of the cell scaffold) to such adegree that they form a substantially unitary structure where cellspresent within the biological tissue may require electricallycommunications with the nanoscale wires in order to function, or tocommunicate with each other. For example, cardiac or muscle cells withina tissue may not be able to beat or contract, or may not be able to beator contract in a regular fashion, without stimuli from the nanoscalewires, or without using the nanoscale wires to communicate. As anotherexample, nerve cells within the tissue may form axons and/or dendriteswith the nanoscale wires, e.g., in order to transmit and/or receiveelectronic signals from other nerve cells and/or from the nanoscalewires. In such fashion, an electrically unitary structure may begenerated, i.e., a “cyborg” tissue can be created whose biologicalfunctioning depends not only on the cells or tissues, but on theelectronic components as well, e.g., such that the distinction betweenthe biological and electronic systems becomes blurred.

In another set of embodiments, the biological tissue may be one thatcontains sufficient nanoscale wires that a property, such as a chemicalor an electrical property, can be determined at a relatively highresolution, and/or in three dimensions within the biological tissue,e.g., due to the placement of nanoscale wires within the tissue that canbe used as sensors. For example, one or more nanoscale wires may bepresent within an electronic circuit as a component of a field effecttransistor. In addition, in certain embodiments, such determinations maybe transmitted and/or recorded, e.g., for later use and or analysis.

Thus, for example, a property such as a chemical property and/or anelectrical property can be determined at a resolution of less than about2 mm, less than about 1 mm, less than about 500 micrometers, less thanabout 300 micrometers, less than about 100 micrometers, less than about50 micrometers, less than about 30 micrometers, or less than about 10micrometers, etc., e.g., due to the average separation between ananoscale wire and its nearest neighboring nanoscale wire. In addition,as mentioned, the property may be determined within the tissue in 3dimensions in some instances, in contrast with many other techniqueswhere only a surface of the biological tissue can be studied.Accordingly, very high resolution and/or 3-dimensional mappings of theproperty of the biological tissue can be obtained in some embodiments.Any suitable tissue may be studied, e.g., cardiac tissue, vasculartissue, muscle, cartilage, bone, liver tissue, pancreatic tissue,bladder tissue, airway tissues, bone marrow tissue, or the like.

In addition, in some cases, such properties can be determined and/orrecorded as a function of time. Thus, for example, such properties canbe determined at a time resolution of less than about 1 min, less thanabout 30 s, less than about 15 s, less than about 10 s, less than about5 s, less than about 3 s, less than about 1 s, less than about 500 ms,less than about 300 ms, less than about 100 ms, less than about 50 ms,less than about 30 ms, less than about 10 ms, less than about 5 ms, lessthan about 3 ms, less than about 1 ms, etc.

In yet another set of embodiments, the biological tissue, and/orportions of the biological tissue, may be electrically stimulated usingnanoscale wires present within the tissue. For example, all, or a subsetof the electrically active nanoscale wires may be electricallystimulated, e.g., by using an external electrical system, such as acomputer. Thus, for example, a single nanoscale wire, a group ofnanoscale wires, or substantially all of the nanoscale wires can beelectrically stimulated, depending on the particular application. Insome cases, such nanoscale wires can be stimulated in a particularpattern, e.g., to cause cardiac or muscle cells to contract or beat in aparticular pattern (for example, as part of a prosthetic or apacemaker), to cause the firing of neurons with a particular pattern, tomonitor the status of an implanted tissue within a subject, or the like.

Another aspect of the present invention is generally directed to systemsand methods for making and using such cell scaffolds. Briefly, in oneset of embodiments, a scaffold is constructed by assembling variouspolymers, metals, nanoscale wires, and other components together on asubstrate. For example, lithographic techniques such as e-beamlithography, photolithography, X-ray lithography, extreme ultravioletlithography, ion projection lithography, etc. may be used to patternpolymers, metals, etc. on the substrate, and nanoscale wires can beprepared separately then added to the substrate. After assembly, atleast a portion of the substrate (e.g., a sacrificial material) may beremoved, allowing the scaffold to be partially or completely removedfrom the substrate. The scaffold can, in some cases, be formed into a3-dimensional structure, for example, spontaneously, or by folding orrolling the structure. Other materials may also be added to thescaffold, e.g., to help stabilize the structure, to add additionalagents to enhance its biocompatibility, etc. The scaffold can be used invivo, e.g., by implanting it in a subject, and/or in vitro, e.g., byseeding cells, etc. on the scaffold. In addition, in some cases, cellsmay initially be grown on the scaffold before the scaffold is implantedinto a subject. A schematic diagram of the layers formed on thesubstrate in one embodiment is shown in FIG. 2. However, it should beunderstood that this diagram is illustrative only and is not drawn toscale, and not all of the layers shown in FIG. 2 are necessarilyrequired in every embodiment of the invention.

The substrate (200 in FIG. 2) may be chosen to be one that can be usedfor lithographic techniques such as e-beam lithography orphotolithography, or other lithographic techniques including thosediscussed herein. For example, the substrate may comprise or consistessentially of a semiconductor material such as silicon, although othersubstrate materials (e.g., a metal) can also be used. Typically, thesubstrate is one that is substantially planar, e.g., so that polymers,metals, and the like can be patterned on the substrate.

In some cases, a portion of the substrate can be oxidized, e.g., formingSiO₂ and/or Si₃N₄ on a portion of the substrate, which may facilitatesubsequent addition of materials (metals, polymers, etc.) to thesubstrate. In some cases, the oxidized portion may form a layer ofmaterial on the substrate (205 in FIG. 2), e.g., having a thickness ofless than about 5 micrometers, less than about 4 micrometers, less thanabout 3 micrometers, less than about 2 micrometers, less than about 1micrometer, less than about 900 nm, less than about 800 nm, less thanabout 700 nm, less than about 600 nm, less than about 500 nm, less thanabout 400 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm, etc.

In certain embodiments, one or more polymers can also be deposited orotherwise formed prior to depositing the sacrificial material. In somecases, the polymers may be deposited or otherwise formed as a layer ofmaterial (210 in FIG. 2) on the substrate. Deposition may be performedusing any suitable technique, e.g., using lithographic techniques suchas e-beam lithography, photolithography, X-ray lithography, extremeultraviolet lithography, ion projection lithography, etc. In some cases,some or all of the polymers may be biocompatible and/or biodegradable.The polymers that are deposited may also comprise methyl methacrylateand/or poly(methyl methacrylate), in some embodiments. One, two, or morelayers of polymer can be deposited (e.g., sequentially) in variousembodiments, and each layer may independently have a thickness of lessthan about 5 micrometers, less than about 4 micrometers, less than about3 micrometers, less than about 2 micrometers, less than about 1micrometer, less than about 900 nm, less than about 800 nm, less thanabout 700 nm, less than about 600 nm, less than about 500 nm, less thanabout 400 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm, etc.

Next, a sacrificial material may be deposited. The sacrificial materialcan be chosen to be one that can be removed without substantiallyaltering other materials (e.g., polymers, other metals, nanoscale wires,etc.) deposited thereon. For example, in one embodiment, the sacrificialmaterial may be a metal, e.g., one that is easily etchable. Forinstance, the sacrificial material can comprise germanium or nickel,which can be etched or otherwise removed, for example, using a peroxide(e.g., H₂O₂) or a nickel etchant (many of which are readily availablecommercially). In some cases, the sacrificial material may be depositedon oxidized portions or polymers previously deposited on the substrate.In some cases, the sacrificial material is deposited as a layer (e.g.,215 in FIG. 2). The layer can have a thickness of less than about 5micrometers, less than about 4 micrometers, less than about 3micrometers, less than about 2 micrometers, less than about 1micrometer, less than about 900 nm, less than about 800 nm, less thanabout 700 nm, less than about 600 nm, less than about 500 nm, less thanabout 400 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm, etc.

In some embodiments, a “bedding” polymer can be deposited, e.g., on thesacrificial material. The bedding polymer may include one or morepolymers, which may be deposited as one or more layers (220 in FIG. 2).The bedding polymer can be used to support the nanoscale wires, and insome cases, partially or completely surround the nanoscale wires,depending on the application. For example, as discussed below, one ormore nanoscale wires may be deposited on at least a portion of theuppermost layer of bedding polymer.

For instance, the bedding polymer can at least partially define a cellscaffold. In one set of embodiments, the bedding polymer may bedeposited as a layer of material, such that portions of the beddingpolymer may be subsequently removed. For example, the bedding polymercan be deposited using lithographic techniques such as e-beamlithography, photolithography, X-ray lithography, extreme ultravioletlithography, ion projection lithography, etc., or using other techniquesfor removing polymer that are known to those of ordinary skill in theart. In some cases, more than one bedding polymer is used, e.g.,deposited as more than one layer (e.g., sequentially), and each layermay independently have a thickness of less than about 5 micrometers,less than about 4 micrometers, less than about 3 micrometers, less thanabout 2 micrometers, less than about 1 micrometer, less than about 900nm, less than about 800 nm, less than about 700 nm, less than about 600nm, less than about 500 nm, less than about 400 nm, less than about 300nm, less than about 200 nm, less than about 100 nm, etc. For example, insome embodiments, portions of the photoresist may be exposed to light(visible, UV, etc.), electrons, ions, X-rays, etc. (e.g., projected ontothe photoresist), and the exposed portions can be etched away (e.g.,using suitable etchants, plasma, etc.) to produce the pattern.

Accordingly, the bedding polymer may be formed into a particularpattern, e.g., in a grid, or in a pattern that suggests an endogenouscell scaffold, before or after deposition of nanoscale wires (asdiscussed in detail below), in certain embodiments of the invention. Thepattern can be regular or irregular. For example, the bedding polymercan be formed into a pattern defining pore sizes such as those discussedherein. For instance, the polymer may have an average pore size of atleast about 100 micrometers, at least about 200 micrometers, at leastabout 300 micrometers, at least about 400 micrometers, at least about500 micrometers, at least about 600 micrometers, at least about 700micrometers, at least about 800 micrometers, at least about 900micrometers, or at least about 1 mm, and/or an average pore size of nomore than about 1.5 mm, no more than about 1.4 mm, no more than about1.3 mm, no more than about 1.2 mm, no more than about 1.1 mm, no morethan about 1 mm, no more than about 900 micrometers, no more than about800 micrometers, no more than about 700 micrometers, no more than about600 micrometers, or no more than about 500 micrometers, etc.

Any suitable polymer may be used as the bedding polymer. In some cases,one or more of the polymers can be chosen to be biocompatible and/orbiodegradable. In certain embodiments, one or more of the beddingpolymers may comprise a photoresist. Photoresists can be useful due totheir familiarity in use in lithographic techniques such as thosediscussed herein. Non-limiting examples of photoresists include SU-8,S1805, LOR 3A, poly(methyl methacrylate), poly(methyl glutarimide),phenol formaldehyde resin (diazonaphthoquinone/novolac),diazonaphthoquinone (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley1400-17, Shipley 1400-27, Shipley 1400-37, etc., as well as any othersdiscussed herein.

In certain embodiments, one or more of the bedding polymers can beheated or baked, e.g., before or after depositing nanoscale wiresthereon as discussed below, and/or before or after patterning thebedding polymer. For example, such heating or baking, in some cases, isimportant to prepare the polymer for lithographic patterning. In variousembodiments, the bedding polymer may be heated to a temperature of atleast about 30° C., at least about 65° C., at least about 95° C., atleast about 150° C., or at least about 180° C., etc.

Next, one or more nanoscale wires (e.g., 225 in FIG. 2) may bedeposited, e.g., on a bedding polymer on the substrate. Any of thenanoscale wires described herein may be used, e.g., n-type and/or p-typenanoscale wires, substantially uniform nanoscale wires (e.g., having avariation in average diameter of less than 20%), nanoscale wires havinga diameter of less than about 1 micrometer, semiconductor nanowires,silicon nanowires, bent nanoscale wires, kinked nanoscale wires,core/shell nanowires, nanoscale wires with heterojunctions, etc. In somecases, the nanoscale wires are present in a liquid which is applied tothe substrate, e.g., poured, painted, or otherwise deposited thereon. Insome embodiments, the liquid is chosen to be relatively volatile, suchthat some or all of the liquid can be removed by allowing it tosubstantially evaporate, thereby depositing the nanoscale wires. In somecases, at least a portion of the liquid can be dried off, e.g., byapplying heat to the liquid. Examples of suitable liquids include wateror isopropanol.

In some cases, at least some of the nanoscale wires may be at leastpartially aligned, e.g., as part of the deposition process, and/or afterthe nanoscale wires have been deposited on the substrate. Thus, thealignment can occur before or after drying or other removal of theliquid, if a liquid is used. Any suitable technique may be used foralignment of the nanoscale wires. For example, the nanoscale wires canbe aligned by passing or sliding substrates containing the nanoscalewires past each other (see, e.g., International Patent Application No.PCT/US2007/008540, filed Apr. 6, 2007, entitled “Nanoscale Wire Methodsand Devices,” by Nam, et al., published as WO 2007/145701 on Dec. 21,2007, incorporated herein by reference in its entirety), the nanoscalewires can be aligned using Langmuir-Blodgett techniques (see, e.g., U.S.patent application Ser. No. 10/995,075, filed Nov. 22, 2004, entitled“Nanoscale Arrays and Related Devices,” by Whang, et al., published asU.S. Patent Application Publication No. 2005/0253137 on Nov. 17, 2005,incorporated herein by reference in its entirety), the nanoscale wirescan be aligned by incorporating the nanoscale wires in a liquid film or“bubble” which is deposited on the substrate (see, e.g., U.S. patentapplication Ser. No. 12/311,667, filed Apr. 8, 2009, entitled “LiquidFilms Containing Nanostructured Materials,” by Lieber, et al., publishedas U.S. Patent Application Publication No. 2010/0143582 on Jun. 10,2010, incorporated by reference herein in its entirety), or a gas orliquid can be passed across the nanoscale wires to align the nanoscalewires (see, e.g., U.S. Pat. No. 7,211,464, issued May 1, 2007, entitled“Doped Elongated Semiconductors, Growing Such Semiconductors, DevicesIncluding Such Semiconductors, and Fabricating Such Devices,” by Lieber,et al.; and U.S. Pat. No. 7,301,199, issued Nov. 27, 2007, entitled“Nanoscale Wires and Related Devices,” by Lieber, et al., eachincorporated herein by reference in its entirety). Combinations of theseand/or other techniques can also be used in certain instances. In somecases, the gas may comprise an inert gas and/or a noble gas, such asnitrogen or argon.

In certain embodiments, a “lead” polymer is deposited (230 in FIG. 2),e.g., on the sacrificial material and/or on at least some of thenanoscale wires. The lead polymer may include one or more polymers,which may be deposited as one or more layers. The lead polymer can beused to cover or protect metal leads or other conductive pathways, whichmay be subsequently deposited on the lead polymer. In some embodiments,the lead polymer can be deposited, e.g., as a layer of material suchthat portions of the lead polymer can be subsequently removed, forinstance, using lithographic techniques such as e-beam lithography,photolithography, X-ray lithography, extreme ultraviolet lithography,ion projection lithography, etc., or using other techniques for removingpolymer that are known to those of ordinary skill in the art, similar tothe bedding polymers previously discussed. However, the lead polymersneed not be the same as the bedding polymers (although they can be), andthey need not be deposited using the same techniques (although they canbe). In some cases, more than one lead polymer may be used, e.g.,deposited as more than one layer (for example, sequentially), and eachlayer may independently have a thickness of less than about 5micrometers, less than about 4 micrometers, less than about 3micrometers, less than about 2 micrometers, less than about 1micrometer, less than about 900 nm, less than about 800 nm, less thanabout 700 nm, less than about 600 nm, less than about 500 nm, less thanabout 400 nm, less than about 300 nm, less than about 200 nm, less thanabout 100 nm, etc.

Any suitable polymer can be used as the lead polymer. In some cases, oneor more of the polymers may be chosen to be biocompatible and/orbiodegradable. For example, in one set of embodiments, one or more ofthe polymers may comprise poly(methyl methacrylate). In certainembodiments, one or more of the lead polymers comprises a photoresist,such as those described herein.

In certain embodiments, one or more of the lead polymers may be heatedor baked, e.g., before or after depositing nanoscale wires thereon asdiscussed below, and/or before or after patterning the lead polymer. Forexample, such heating or baking, in some cases, is important to preparethe polymer for lithographic patterning. In various embodiments, thelead polymer may be heated to a temperature of at least about 30° C., atleast about 65° C., at least about 95° C., at least about 150° C., or atleast about 180° C., etc.

Next, a metal or other conductive material can be deposited (235 in FIG.2), e.g., on one or more of the lead polymer, the sacrificial material,the nanoscale wires, etc. to form a metal lead or other conductivepathway. More than one metal can be used, which may be deposited as oneor more layers. For example, a first metal may be deposited, e.g., onone or more of the lead polymers, and a second metal may be deposited onat least a portion of the first metal. Optionally, more metals can beused, e.g., a third metal may be deposited on at least a portion of thesecond metal, and the third metal may be the same or different from thefirst metal. In some cases, each metal may independently have athickness of less than about 5 micrometers, less than about 4micrometers, less than about 3 micrometers, less than about 2micrometers, less than about 1 micrometer, less than about 900 nm, lessthan about 800 nm, less than about 700 nm, less than about 600 nm, lessthan about 500 nm, less than about 400 nm, less than about 300 nm, lessthan about 200 nm, less than about 100 nm, less than about 80 nm, lessthan about 60 nm, less than about 40 nm, less than about 30 nm, lessthan about 20 nm, less than about 10 nm, less than about 8 nm, less thanabout 6 nm, less than about 4 nm, or less than about 2 nm, etc., and thelayers may be of the same or different thicknesses.

Any suitable technique can be used for depositing metals, and if morethan one metal is used, the techniques for depositing each of the metalsmay independently be the same or different. For example, in one set ofembodiments, deposition techniques such as sputtering can be used. Otherexamples include, but are not limited to, physical vapor deposition,vacuum deposition, chemical vapor deposition, cathodic arc deposition,evaporative deposition, e-beam PVD, pulsed laser deposition, ion-beamsputtering, reactive sputtering, ion-assisted deposition,high-target-utilization sputtering, high-power impulse magnetronsputtering, gas flow sputtering, or the like.

The metals can be chosen in some cases such that the deposition processyields a pre-stressed arrangement, e.g., due to atomic lattice mismatch,which causes the subsequent metal leads to warp or bend, for example,once released from the substrate. Although such processes were typicallyundesired in the prior art, in certain embodiments of the presentinvention, such pre-stressed arrangements may be used to cause theresulting cell scaffold to form a 3-dimensional structure, in some casesspontaneously, upon release from the substrate. However, it should beunderstood that in other embodiments, the metals may not necessary bedeposited in a pre-stressed arrangement.

Examples of metals that can be deposited (stressed or unstressed)include, but are not limited to, aluminum, gold, silver, copper,molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium,as well as any combinations of these and/or other metals. For example, achromium/palladium/chromium deposition process, in some embodiments, mayform a pre-stressed arrangement that is able to spontaneously form a3-dimensional structure after release from the substrate.

In certain embodiments, a “coating” polymer can be deposited (240 inFIG. 2), e.g., on at least some of the conductive pathways and/or atleast some of the nanoscale wires. The coating polymer may include oneor more polymers, which may be deposited as one or more layers. In someembodiments, the coating polymer may be deposited on one or moreportions of a substrate, e.g., as a layer of material such that portionsof the coating polymer can be subsequently removed, e.g., usinglithographic techniques such as e-beam lithography, photolithography,X-ray lithography, extreme ultraviolet lithography, ion projectionlithography, etc., or using other techniques for removing polymer thatare known to those of ordinary skill in the art, similar to the otherpolymers previously discussed. The coating polymers can be the same ordifferent from the lead polymers and/or the bedding polymers. In somecases, more than one coating polymer may be used, e.g., deposited asmore than one layer (e.g., sequentially), and each layer mayindependently have a thickness of less than about 5 micrometers, lessthan about 4 micrometers, less than about 3 micrometers, less than about2 micrometers, less than about 1 micrometer, less than about 900 nm,less than about 800 nm, less than about 700 nm, less than about 600 nm,less than about 500 nm, less than about 400 nm, less than about 300 nm,less than about 200 nm, less than about 100 nm, etc.

Any suitable polymer may be used as the coating polymer. In some cases,one or more of the polymers can be chosen to be biocompatible and/orbiodegradable. For example, in one set of embodiments, one or more ofthe polymers may comprise poly(methyl methacrylate). In certainembodiments, one or more of the coating polymers may comprise aphotoresist, e.g., as discussed herein.

In certain embodiments, one or more of the coating polymers can beheated or baked, e.g., before or after depositing nanoscale wiresthereon as discussed below, and/or before or after patterning thecoating polymer. For example, such heating or baking, in some cases, isimportant to prepare the polymer for lithographic patterning. In variousembodiments, the coating polymer may be heated to a temperature of atleast about 30° C., at least about 65° C., at least about 95° C., atleast about 150° C., or at least about 180° C., etc.

After formation of the cell scaffold, some or all of the sacrificialmaterial may then be removed in some cases. In one set of embodiments,for example, at least a portion of the sacrificial material is exposedto an etchant able to remove the sacrificial material. For example, ifthe sacrificial material is a metal such as nickel, a suitable etchant(for example, a metal etchant such as a nickel etchant, acetone, etc.)can be used to remove the sacrificial metal. Many such etchants may bereadily obtained commercially. In addition, in some embodiments, thecell scaffold can also be dried, e.g., in air (e.g., passively), byusing a heat source, by using a critical point dryer, etc.

In certain embodiments, upon removal of the sacrificial material,pre-stressed portions of the cell scaffold (e.g., metal leads containingdissimilar metals) can spontaneously cause the cell scaffold to adopt a3-dimensional structure. In some cases, the cell scaffold may form a3-dimensional structure as discussed herein. For example, the cellscaffold may have an open porosity of at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 97, at least about 99%, at leastabout 99.5%, or at least about 99.8%. The cell scaffold may also have,in some cases, an average pore size of at least about 100 micrometers,at least about 200 micrometers, at least about 300 micrometers, at leastabout 400 micrometers, at least about 500 micrometers, at least about600 micrometers, at least about 700 micrometers, at least about 800micrometers, at least about 900 micrometers, or at least about 1 mm,and/or an average pore size of no more than about 1.5 mm, no more thanabout 1.4 mm, no more than about 1.3 mm, no more than about 1.2 mm, nomore than about 1.1 mm, no more than about 1 mm, no more than about 900micrometers, no more than about 800 micrometers, no more than about 700micrometers, no more than about 600 micrometers, or no more than about500 micrometers, etc.

However, in other embodiments, further manipulation may be needed tocause the cell scaffold to adopt a 3-dimensional structure, e.g., onewith properties such as is discussed herein. For example, after removalof the sacrificial material, the cell scaffold may need to be rolled,curled, folded, creased, etc., or otherwise manipulated to form the3-dimesional structure. Such manipulations can be done using anysuitable technique, e.g., manually, or using a machine.

Other materials may be also added to the cell scaffold, e.g., before orafter it forms a 3-dimensional structure, for example, to help stabilizethe structure, to add additional agents to enhance its biocompatibility(e.g., growth hormones, extracellular matrix protein, Matrigel™, etc.),to cause it to form a suitable 3-dimension structure, to control poresizes, etc. Non-limiting examples of such materials have been previouslydiscussed above, and include other polymers, growth hormones,extracellular matrix protein, specific metabolites or nutrients,additional scaffold materials, or the like.

In addition, in some cases, cells are plated or seeded on the cellscaffold and allowed to grow. For example, the cells may be plated onthe cell scaffold in vitro, and/or the cell scaffold may be exposed oreven submerged within a suitable cell growth medium. Such media arewidely available commercially. In some embodiments, the cell scaffoldcan be subsequently implanted in vivo into a subject, e.g., upon thegrowth of suitable from the cells. In other cases, the cell scaffold candirectly be used in an in vivo setting, i.e., without needing plating ofcells, and/or without formation of tissues before implantation.

In addition, the cell scaffold can be interfaced in some embodimentswith one or more electronics, e.g., an external electrical system suchas a computer or a transmitter (for instance, a radio transmitter, awireless transmitter, etc.). In some cases, electronic testing of thecell scaffold may be performed, e.g., before or after implantation intoa subject. For instance, one or more of the metal leads may be connectedto an external electrical circuit, e.g., to electronically interrogateor otherwise determine the electronic state or one or more of thenanoscale wires within the cell scaffold. Such determinations may beperformed quantitatively and/or qualitatively, depending on theapplication, and can involve all, or only a subset, of the nanoscalewires contained within the cell scaffold, e.g., as discussed herein.

The following documents are incorporated herein by reference: U.S. Pat.No. 7,211,464, issued May 1, 2007, entitled “Doped ElongatedSemiconductors, Growing Such Semiconductors, Devices Including SuchSemiconductors, and Fabricating Such Devices,” by Lieber, et al.; U.S.Pat. No. 7,301,199, issued Nov. 27, 2007, entitled “Nanoscale Wires andRelated Devices,” by Lieber, et al.; and International PatentApplication No. PCT/US2010/050199, filed Sep. 24, 2010, entitled “BentNanowires and Related Probing of Species,” by Tian, et al., published asWO 2011/038228 on Mar. 31, 2011.

In addition, incorporated herein by reference is a U.S. provisionalapplication, filed on even date herewith, entitled “Scaffolds ComprisingNanoelectronic Components for Cells, Tissue, and Other Applications,” byLieber, et al. Also incorporated herein by reference in their entiretiesare U.S. Prov. Pat. Apl. Ser. No. 61/698,492, entitled “Methods AndSystems For Scaffolds Comprising Nanoelectronic Components,” filed Sep.7, 2012, and U.S. Prov. Pat. Apl. Ser. No. 61/698,502, entitled“Scaffolds Comprising Nanoelectronic Components For Cells, Tissues, AndOther Applications,” filed Sep. 7, 2012.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

The development of three-dimensional (3D) synthetic biomaterials asstructural and bioactive extracellular matrices (ECMs) is central tofields ranging from cellular biophysics to regenerative medicine. As ofyet, it has not been possible to provide spatiotemporal monitoring ofcells throughout 3D scaffolds, although this capability could have amarked impact. This example illustrates a new platform of seamlesslyintegrating nanoelectronic devices into free-standing, flexible, andbiocompatible nanoelectronic scaffolds (nanoES) for 3D cell and tissueapplications.

The scaffolds in this and the following examples were prepared by planarlithography with nanowire transistors serving as sensor elements andmetal interconnects sandwiched between biocompatible polymeric scaffoldnetworks. 3D macroporous scaffold structures were formed either byself-organization of coplanar reticular networks with built-in strain orby manual folding or rolling of 2D mesh matrices. The scaffoldsexhibited robust electronic properties during conversion from planarnetworks to 3D structures, and were used as extracellular scaffolds forefficient 3D culture of neurons, cardiomyocytes and smooth muscle cells.Notably, multiplexed electrical recordings of extracellular potentialsfrom 3D innervated cardiac patches demonstrated the feasibility ofcontinuous monitoring in 3D of excitation propagation. 3D distributednanoelectronic devices were also used for simultaneous monitoring of pHinside and outside tubular vascular smooth muscle constructs. Thisapproach allows functionalizing engineered tissues, indwelling 3Dtissue-based therapeutic assays, enhanced biomedical prosthetics, andmakes possible novel biomaterials/biosystems where the distinctionbetween biological and electronic systems becomes blurred.

This approach integrates nanoelectronics into tissues in 3D. Siliconnanowire field-effect transistor-based nanoelectronic biomaterials wereused given their capability for recording both extracellular andintracellular signals with subcellular resolution. This design (FIG. 3)involved stepwise incorporation of biomimetic and biological elementsinto nanoelectronic networks across nanometer to centimeter size scales.First, chemically synthesized kinked and/or uniform silicon nanowireswere deposited either randomly or in regular patterns forsingle-nanowire FETs (step A, FIG. 3), forming the nanoelectronic sensorelements of the hybrid biomaterials. Second, individual nanowirefield-effect transistor (NWFET) devices were lithographically patternedand integrated into free-standing macroporous scaffolds (step B, FIG.3), termed “nanoelectronic scaffolds (nanoES).” The nanoES were tailoredto be 3D, to have nanometer to micrometer features with high (>99%)porosity, and to be highly flexible and biocompatible. NanoES could alsobe hybridized with biodegradable synthetic or natural macroporous ECMsproviding ECMs with electrical sensory function and nanoES withbiochemical environments suitable for tissue culture. Finally, cellswere cultured on nanoES (step C, FIG. 3).

In particular, FIG. 3 schematically illustrates that conventional bulkelectronics (left column) are distinct from biological systems (rightcolumn) in composition, structural hierarchy, mechanics and function.Their electrical coupling at the tissue/organ level is usually limitedto the tissue surface, where only boundary or global information can begleaned unless invasive approaches are used. The present example, incontrast, demonstrates an integrated system from the discrete buildingblocks of electronic and biological systems, e.g., semiconductornanowires, molecular precursors of polymers and single cells. Thesebiomimetic and bottom-up steps are used: A) patterning, metallizationand epoxy passivation to form single NWFETs, B) forming 3D NWFETsmatrices (nanoelectric scaffolds) by self- or manual organization andhybridization with synthetic biomaterials, and C) incorporation of cellsand growth of synthetic tissue via biological processes. In thesefigures, the circles represent nanowire components, and the ribbonsrepresent metal and epoxy interconnects (darker ribbons), andtraditional extracellular matrices (lighter ribbons).

As examples, two different types of nanoESs (FIG. 4A) were designed thatwere free-standing, flexible and contained similar components. Both werefabricated on sacrificial layers, which were subsequently removed,yielding free-standing nanoES. These will be discussed in greater detailbelow. In brief, a layer of resist (SU-8) was coated on a sacrificiallayer of material, nanowires were deposited, the scaffold structure waspatterned with lithography, metal interconnections were defined bylithography and deposition, a second layer of resist (SU-8) was coatedthereon, and lithography was used to define this as the upper layer ofpassivation over the interconnects.

One type of nanoES was termed a “reticular” nanoES. The reticularnanoESs were made by electron beam lithography. Self-organizationcreated a random or regular network of 3D features that mimiced the sizescale and morphology of submicron ECM features, like the fibrousmeshwork of brain ECM. The other type was called a “mesh” nanoES. Themesh nanoESs were made by photolithography with a regular structure,like the ECM of the ventricular myocardium. 3D aspects were created byrolling or folding these structures to form a 3D scaffold. 3D scaffoldswere then realized in a straightforward manner by directed meshmanipulation. The planar design and initial fabrication of these 3DnanoES used existing capabilities similar to those developed forconventional planar nanoelectronics, and allow integration of additionaldevice components (for example, memories and logic gates) or substantialincreases in device number or overall scaffold size.

The 2D structure of the reticular scaffold was designed so that metalinterconnects were stressed. Removal of the sacrificial layer promptedself-organization into 3D. Reconstructed 3D confocal fluorescence imagesof a typical reticular nanoES viewed along y- and x-axes (FIG. 4B,Panels I and II respectively) showed that the framework was 3D with ahighly curvilinear and interconnected structure consistent with thedesign (FIG. 4A, Panel I). The porosity (calculated from the initialplanar device design and the final 3D construct volume) was greater than99.8%, comparable to that of hydrogel biomaterials. NWFET devices (FIG.4B, Panel II) within the scaffold spanned separations of 7.3 to 324micrometers in 3D (FIG. 5), and the reticular scaffold heights were lessthan about 300 micrometers for these fabrication conditions. Inaddition, the devices could also be made closer together (for example,less than 0.5 micrometers), e.g., by depositing the nanowires moredensely on the substrate, for instance, to improve the spatialresolution of nanoelectronic sensors; the span of device separations andscaffold heights can also be increased substantially using larger fieldlithography. FIG. 5 shows a NWFET 3D distribution in fibrous nanoES. 14NWFETs were distributed in the construct shown in FIG. 4B. Individualdevices are shown as solid spheres. The overall size of the scaffold,x-y-z was ˜300-400-200 micrometers. The NWFET devices within thescaffold were separated in 3 dimensions by 7.3 micrometers to 324micrometers.

Scanning electron microscopy (SEM) of the reticular nanoES (FIG. 4C)revealed kinked nanowires (about 80 nm in diameter), and metallicinterconnects (about 0.7 micrometers in diameter) contained within anSU-8 backbone (about 1 micrometer in width). The feature sizes werecomparable to those of synthetic and natural ECMs, and were severalorders of magnitude smaller than those of reported electronic structurespenetrating tissue in 3D. Water-gate measurements of the NWFET elementsof the 3D scaffolds in aqueous medium (see below) demonstrated deviceyields of ˜80%, conductances of 1.52+/−0.61 microsiemens (mean+/−SD) andsensitivities of 8.07+/−2.92 microsiemens/V, comparable to measurementsfrom planar devices using similar nanowires.

The 3D mesh nanoES were prepared by manual folding and rolling offree-standing device arrays. The mesh structures (FIG. 4A, Panel II)were fabricated such that the nanoES maintained an approximately planarconfiguration following relief from the fabrication substrate (seebelow). A typical 3.5 cm×1.5 cm×˜2 micrometer mesh nanoES (FIG. 4D), wassubstantially planar with 60 addressable NWFET devices distributed in aregular array (FIG. 4D) and had a 2D open porosity of 75% (FIG. 4D). Themesh porosity was comparable to that of honeycomb-like synthetic ECMengineered for cardiac tissue culture. The nanowires (FIG. 4D1), metalinterconnects (FIG. 4D2), and SU-8 structural elements (FIG. 4D3) had anareal mass density of less than 60 micrograms/cm². The mesh nanoES washighly flexible and could be manually rolled into tubular 3D constructswith inner diameters at least as small as 1.5 mm (FIG. 4E), and folded.Macroporous structures of the open mesh nanoES were formed either byloosely stacking adjacent mesh layers (FIG. 4F) or by shaping it withother biomaterials. These capabilities were consistent with theestimated ultralow effective bending stiffness, which was tuned between0.006 and 1.3 nNm for this mesh and was comparable to planar epidermalelectronics.

The electrical transport characteristics of the mesh nanoES wasevaluated in phosphate buffered saline (PBS. The typical device yieldwas 90-97%, with average device conductances of ˜3 microsiemens andsensitivity of ˜7 microsiemens per volt (FIG. 4G). Representative data(FIG. 4H) from single NWFET (FIG. 4H, light dots in upper panel) showeda less than 0.17 microsiemens conductance change (ΔG) or less than 2.3%total change for 6 revolutions. The device sensitivity (S) remainedstable with a maximum change (ΔS) of 0.031 microsiemens/V, or a 1.5%variation. The stable device performance can be explained by the lowestimated strains of metal (less than 0.005%) and SU-8 (less than 0.27%)layers in this tubular construct (see below), and showed that electricaltransport properties were substantially independent of location.Furthermore, 14 devices evenly distributed on 6 layers of a fullyrolled-up tubular scaffold (FIG. 4I) showed a maximum conductance change(ΔG) of 6.8% and a maximum change in device sensitivity (ΔS) of 6.9%,versus the initial unrolled state, indicating robust NWFETs. Repetitiverolling and relaxation to the flat state did not degrade NWFETperformance. These findings suggested reliable sensing/recording ofthese dynamic and deformable systems.

Additional details regarding FIG. 4 follow. FIG. 4A illustrates devicefabrication schematics, in accordance to one embodiment of theinvention, for both reticular NWFET devices (Panel I) and mesh NWFETdevices (Panel II). In FIG. 4A, the dots represent individual NWFETs.The outer, larger square (lighter color) represents the silicon oxidesubstrate, while the inner, smaller square (darker color) represents thenickel sacrificial layers. The ribbons on the right of each figurerepresents the nanoES scaffold constructs that are formed. FIG. 4B shows3D reconstructed confocal fluorescence images of reticular nanoES viewedalong the y (Panel I) and x (Panel II) axes. The scaffold was labeledwith rhodamine 6G. The overall size of the structure (x-y-z) was300-400-200 micrometers. The solid and dashed open boxes indicate twoNWFET devices located on different planes along x axis. The scale barsare 20 micrometers. FIG. 4C is an SEM image of a single kinked NWFETwithin a reticular scaffold, showing (1) kinked nanowires, (2) metallicinterconnects (lines) and (3) the SU-8 backbone construct. The scale baris 2 micrometers. FIG. 4D shows a photograph of a mesh device showing(1) nanowires, (2) metal interconnects, and (3) SU-8 structuralelements. The circle indicates the position of a single NWFET. The scalebar is 2 mm. FIG. 4E is a photograph of a partially rolled-up meshdevice. The scale bar is 5 mm. FIG. 4F is a SEM image of a looselypacked mesh nanoES, showing the macroporous structure. The scale bar is100 micrometers. FIG. 4G is a histogram of nanowire FET conductance andsensitivity in one typical mesh nanoES. The conductance and sensitivitywere measured in the water-gate configuration without rolling. Thedevice yield for this mesh nanoES was 95%. FIG. 4H shows water-gatesensitivity and conductance of a NWFET device during the rolling processin a mesh device. The upper panel of FIG. 4H is a schematic of theposition of a NWFET (dot) during rolling process; 0-6 denote the numberof turns. FIG. 4I shows the relative change in conductance andsensitivity of 14 NWFETs evenly distributed throughout a fully rolled-upmesh device. The upper panel is a schematic of the NFWET position(dots). In both FIGS. 4H and 4I, the thicknesses of the tubularstructures have been exaggerated for schematic clarity.

Example 2

Simulations of a subunit of the self-organizing reticular structure wereperformed (FIG. 6A-C) in this example. Measurements of bending for thecorresponding experimental structures (FIG. 6C, open squares) were foundto be consistent with the simulations (FIG. 6C). Additionally, changesin structural parameters (for example, the total length of the subunitand thicknesses of SU-8 or metals) yielded predictable changes in thebending angle of the subunit (FIG. 7). This example thus shows thatordered 3D nanowire FET arrays could be designed and fabricated usingreticular- or mesh-like structures that incorporated multi-layer metalinterconnects with built-in stress to self-organize (roll-up) thescaffold (FIG. 7).

In addition, in some experiments, reticular domains were designed inmesh-like structures (FIG. 6D). Images of reticular domains (FIGS. 6Eand 6F) showed that regular nanowire FET devices with distinct devicepositions could be realized, for example, by varying the structuralparameters of individual elements. Overall, this approach yieldedhierarchical 3D nanoES with submicrometer to micrometer scale control inreticular domains and millimeter to centimeter scale in the mesh matrixby folding or rolling as shown above (FIG. 4).

The reticular and mesh nanoES were also merged with conventionalmacroporous biomaterials in some of these experiments. Specifically, gelcasting, lyophilization and electrospinning were used to deposit andconstruct macroporous collagen (FIG. 8A), alginate (FIG. 8B) andpoly(lactic-co-glycolic acid) (PLGA; FIG. 8C), respectively, aroundnanoES structures. A confocal fluorescence micrograph of a hybridreticular nanoES/collagen scaffold (FIG. 8A) showed that the collagennanofibers (arrow) were fully entangled with the nanoES, with noapparent phase separation.

SEM images of the open mesh nanoES/alginate hybrid scaffold produced bylyophilization (FIG. 8B) showed that the flexible nanoES mesh wasintimately anchored to the alginate framework, which had a similar porestructure as the pure alginate scaffold prepared under similarconditions. Optical micrographs of a multilayered mesh nanoES/PLGAscaffold (FIG. 8C), which was prepared by electrospinning PLGA fibers onboth sides of the nanoES and subsequent folding of the hybrid structure,highlighted the intimate contact between nanoES mesh and PLGA fibers.The hybrid nanoES/biomaterial 3D scaffolds retained the originalnanowire FET device characteristics. For example, measurements in 1×phosphate buffered saline solution showed that ΔG/G and ΔS/S were lessthan +/−9% for the mesh nanoES/PLGA composite versus bare nanoES. Thehybrid nanoES scaffolds were stable under cell culture conditions. Forexample, nanowire FET devices in the hybrid reticular nanoES/Matrigel™scaffold in neuron culture media (FIG. 8D) had ΔS/S less than +/−11%over a nine-week period, showing capability for long-term culture andmonitoring with the nanoES. These results showed that nanoES scaffoldscould be combined with conventional biomaterials to produce hybridscaffolds that provide nanoscale electrical sensory componentsdistributed in three dimensions.

FIGS. 6A and 6B illustrate the basic design and structural subunit forsimulation. In FIG. 6A, a top-down view of the entire subunit is shown.Ribbons are stressed metal lines with SU-8 passivation. Lines are singleSU-8 ribbons without residual stress. FIG. 6B shows a cross-sectionalviews of those two key structural elements used for simulation. FIG. 6Cis a plot of projected (on the x-y plane) length versus height (in the zdirection) for the vertical ribbon in FIG. 6A as determined from thesimulation. Open squares with error bars are experimental data(mean+/−SD) recorded in air for point A and B in FIG. 6A. The simulationof the bending of the subunit model for the reticular structure wascarried out using the commercial finite element software ABAQUS. Theinset shows a 3D view of the simulated structure, and the scale barshows different heights in the z direction.

FIG. 6D is a schematic showing the integration of periodicreticular-device domains (filled rectangles) into a flexible mesh. Inindividual reticular domains, the 3D device positions relative to theglobal flexible mesh could be controlled by their geometry designs (FIG.6A-C). In FIGS. 6E and 6F, design patterns (I) and experimental data(II) for two reticular units are shown. SU-8, metal and nanowires areshown in this figure. In FIG. 6E, changing the structure of theconnecting feature (arrows) between adjacent device units during patterndesign (I) yielded controlled variations in the 3D positioning of thenanowire FETs, which could be further tuned by the stress in the metalconnections. In these experiments, the device positions were 40micrometers (FIG. 6E, panel II) and 23 micrometers (FIG. 6F, panel II)above the mesh plane. The scale bars in FIGS. 6E and 6F are 20micrometers.

In FIG. 7A, the simulation showed that when the equivalent bendingmoment is increased by 10 times, the subunit structure scrolled up onitself. The inset shows the curve of the central vertical ribbon in FIG.6A, demonstrating the devices were scrolled up and different layers wereseparated. A and B are the two points in FIG. 6A. FIGS. 7B-E show thedesign and fabrication of a much larger and regular matrix and thedensity of stressed elements increasing upward (from 1 to 10) in amanner analogous to the simulated subunit. In FIG. 7B, the verticallines indicate stressed metal lines with SU-8 as passivation, thehorizontal lines indicate non-stressed metal lines for interconnectionwith SU-8 as passivation or SU-8 ribbon as framework, and the circlesmark positions for devices. FIG. 7C is a 3D reconstructed confocalfluorescence image showing the side view of the corresponding fabricatedreticular construct following the design in FIG. 7B. The dashed lineshighlight the edge of the scrolled-up reticular nanoES construct. Thewhite numbers and arrows indicate the position of 5 horizontal linescorresponding to those numbered in FIG. 7B. FIGS. 7D and 7E are confocalfluorescence images scanned across the interior of the scaffold atdifferent heights. The images demonstrate that the device regions(circles) located in planes (heights of 80 and 60 micrometers are shown)were aligned, and thus demonstrated the regular arrangement in 3D. Thescale bars in FIGS. 7D and 7E are 50 micrometers. Overall, the resultsshow that larger scale simulations could be used to predict thereticular construct geometry, and allow the self-assembling approach toprovide regular (or irregular) device arrays distributed through 3Dspace by design.

FIG. 8A is a confocal fluorescence micrograph of a hybrid reticularnanoES/collagen matrix. Collagen type-I was stained with fluoresceinisothiocyanate; epoxy ribbons were stained with rhodamine 6G. The whitearrow marks the position of the nanowire. The scale bar is 10micrometers. FIG. 8B is a SEM images of a mesh nanoES/alginate scaffoldwith top (panel I) and side (panel II) views. The scale bars are 200micrometers (panel I) and 100 micrometers (panel II). FIG. 8C is abright-field optical micrograph of the folded scaffold, showingmultilayered structures of PLGA and nanoelectronic interconnects. Theinset shows a photograph of the hybrid sheet before folding. A sheet ofPLGA fibers with diameters of about 1-3 micrometers was deposited onboth sides of the device. No damage or reduction of device yield wasobserved following this deposition. The scale bars are 200 micrometersand 5 mm (inset). FIG. 8D illustrates the relative changes in nanowireFET sensitivity over time in culture (37° C.; 5% CO₂, supplementedneurobasal medium), where n equals 5 and data are mean+/−SD.

Example 3

The reticular and mesh nanoES described in Example 1 were evaluated in3D culture using several types of cells. Embryonic rat hippocampalneurons in Matrigel™ were cultured on the reticular nanoES for 7 to 21days (FIG. 9). Representative 3D reconstructed confocal microscopyimages (FIGS. 10A-B and FIG. 11) from a 2-week culture showed neuronswith a high density of spatially interconnected neurites that penetratedthe reticular nanoES, often passing through the ring-like structuressupporting individual NWFETs (FIGS. 10B and 11 The widths of thescaffold constructs (passivated metal interconnects and structuralribbons) were similar to those of the neurite projections, demonstratingthe merger of electronics with biological systems at an unprecedentedsimilarity in scale.

In one set of experiments, 3D nanoelectronic cardiac culture wasachieved from hybrid mesh nanoES/PLGA scaffolds (FIGS. 12-14). Confocalfluorescence microscopy of a folded cardiac construct (FIG. 10C)revealed a high density of cardiomyocytes in close contact with nanoEScomponents (FIG. 10C). Epi-fluorescence images of cardiac cells on thesurface of the nanoES cardiac patch also showed striationscharacteristic of cardiac tissue (FIG. 10D and FIGS. 13 and 14).

In vitro cytotoxicity of the nanoES was evaluated using 3D neural andcardiac culture systems (FIGS. 10E-F). Differences in hippocampal neuronviability on reticular nanoES/Matrigel™ versus Matrigel™ over 21 dayswere minimal, assessed with a standard live/dead cell assay (FIG. 10E),and between cardiac cells in hybrid mesh nanoES/Matrigel™/PLGA andMatrigel™/PLGA from 2 to 12 days, measured with a metabolic activityassay (FIG. 10F). These studies showed that on the 2-3 week timescale,the nanoES component of the scaffolds has little effect on the cellviability, and thus could be exploited for a number of in vitro studies,including drug screening assays with these synthetic neural and cardiactissues. The main component of nanoES, SU-8, has been demonstrated tohave long-term chronic biocompatibility suitable for in vivo recording.

FIG. 9 shows the chip assembly for neuronal 3D cultures. In FIG. 9A, aNWFET device chip containing a reticular nanoES was cleaned by O₂plasma, and assembled onto a temperature controlled chip carrier. FIG.9B shows a shallow PDMS chamber (dashed box) that was cleaned and placedover the wire-bonded devices. In FIG. 9C, a glass ring was fixed overthe PDMS chamber with silicone elastomer. FIG. 9D shows a gas-permeable,water-impermeable membrane cover that was used for neuron cultureslasting longer than 7 days.

FIGS. 10A-B show 3D reconstructed confocal images of rat hippocampalneurons after 2 week culture in Matrigel™ on reticular nanoES. In thesefigures, neuronal beta-tubulin was stained with Alexa Fluor® 546, andepoxy ribbons were stained with rhodamine 6G. The metal interconnectsare the relatively straight diagonal lines on the right of the image,and are imaged in reflected light mode. Four NWFET devices enclosed inpolymer rings (arrows) can be seen. FIG. 10A: x: 317 micrometers; y: 317micrometers; z: 100 micrometers; FIG. 10B, x: 127 micrometers; y: 127micrometers; z: 68 micrometers. The star in Panel II denotes a neuritepassing through a ring-like structure supporting a NFWET. FIG. 10C showsconfocal fluorescence images of synthetic cardiac patch. In this figure,alpha-actinin of the cardiomyocytes was stained with Alexa Fluor® 488,cell nuclei (circular regions) were stained with Hoechst 34580, and PLGAfibers were stained with rhodamine 6G. Panels II and III are zoomed-inviews of the upper and lower dashed regions of Panel I, showing metalinterconnects, SU-8 scaffold (arrows in Panel II), and electrospun PLGAfibers (arrows in Panel III). Scale bars are 40 micrometers. FIG. 10Dshows an epi-fluorescence image of the surface of the cardiac patch.Alpha-actinin (ribbons) was stained with Alexa Fluor® 488, while cellnuceli (circular regions) were stained with Hoechst 34580. The positionof the source-drain electrodes is outlined with dashed lines; that ofthe nanowire in between them with an arrow. Scale bar is 40 micrometers.FIG. 10E shows the percentage of viable hippocampal neurons cultured innanoES/Matrigel™ vs Matrigel™. Cell viability was evaluated with aLIVE/DEAD cytotoxicity assay. Cells were counted from 3D reconstructedconfocal fluorescence photomicrographs. n=6; data are mean+/−SD. Thedifferences between groups were very small although statisticallysignificant (p<0.05). FIG. 10F shows MTS cytotoxicity assay ofcardiomyocytes evaluated using the MTS assay. n=6; data are mean+/−SD.The differences between groups were very small although statisticallysignificant (p<0.05). FIG. 10G are conductance versus time tracesrecorded from a single-nanowire FET before (top) and after (bottom)applying noradrenaline. FIG. 10H is a multiplex electrical recording ofextracellular field potentials from four nanowire FETs (labeled a-d) ina mesh nanoES. Data are conductance versus time traces of a single spikerecorded at each nanowire FET.

FIG. 11 shows 3D reconstructed confocal fluorescence image of rathippocampal neurons within a reticular nanoES after two weeks inculture. The images show neurons (stained with fluorescent antibodyagainst beta-tubulin) and polymer ribbons (doped with rhodamine 6G dye).The metal interconnects are marked with white arrows, and are imaged inreflected light mode. Dimensions are: x: 127 micrometers; y: 127micrometers; z: 68 micrometers. The images were rotated from the viewshown in FIG. 8B approximately as follows: (left image) 90 degrees aboutthe z-axis, −10 degrees about the y-axis; (right image) 90 degrees aboutthe z-axis, 100 degrees about the y-axis, 40 degrees about the x-axis.Together, these images show that neurites pass through the ringlikestructures supporting individual nanowire FETs.

FIG. 13 shows fluorescence images from the surface of cardiaccell-seeded nanoES, showing alpha-actinin of cardiomyocytes (stainedwith Alexa Fluor® 488 in FIGS. 13A-C), cell nuclei (stained with Hoechst34580 in FIGS. 13A-C) and PLGA fibers (stained with rhodamine 6G inFIGS. 13B-C). Dense cardiomyocyte growth was supported by both nanoES(marked by arrows) in FIG. 13A and electrospun PLGA fibers in hybridPLGA/nanoES in FIG. 13B. FIG. 13C is a zoomed view of the rectangularbox in FIG. 13B, showing (arrows) striated patterns of alpha-actinin.The scale bar is 200 micrometers in FIG. 13A and 20 micrometers in FIG.13B.

FIG. 14A shows an epi-fluorescence image of the cardiac patchhighlighting alpha-actinin (stained with Alexa Fluor® 488) and cellnuclei (stained with Hoechst 34580) of cardiomyocytes. FIG. 14B shows adifferential interference contrast (DIC) image of the same sampleregion, which highlights the S/D electrodes. FIG. 14C shows an overlayof both images to show the positions of S/D electrodes with respect tothe cells (right). The scale bars is 40 micrometers.

Example 4

The monitoring capabilities of nanoES were demonstrated in an innervated3D cardiomyocyte mesh construct (FIG. 10G). The output recorded from asingle-nanowire FET (FIG. 10G) was about 200 micrometers below theconstruct surface showed regularly spaced spikes with a frequency ofabout 1 Hz, a calibrated potential change of about 2-3 mV, asignal/noise greater than or equal to 3 and a width of about 2 ms. Thepeak amplitude, shape, and width were consistent with extracellularrecordings from cardiomyocytes. The potential of the nanoES cardiacpatch to monitor appropriate pharmacological responsiveness (potentiallyconstituting a platform for in vitro pharmacological studies) was alsoinvestigated by dosing it with norepinephrine, a drug that stimulatescardiac contraction via beta-1 (β₁) adrenergic receptors. Measurementsfrom the same nanowire FET device showed a twofold increase in thecontraction frequency following drug application. Interestingly,recordings from two nanowire FETs from the cardiac patch onnoradrenaline application showed submillisecond and millisecond level,heterogeneous cellular responses to the drug (FIG. 15). Additionally,multiplexing measurements made with a reticular nanoES/neural construct(FIG. 16) showed that the 3D response of glutamate activation could bemonitored. Together, these experiments show nanoES constructs canmonitor in vitro the response to drugs from 3D tissue models, and thuscan be used as a platform for in vitro pharmacological studies.

Simultaneous recordings from four nanowire FETs with separations up to6.8 mm in a nanoES/cardiac construct (FIG. 10H) demonstrated multiplexedsensing of a coherently beating cardiac patch, with submillisecond timeresolution. The device in this example yielded a relatively sparsedevice distribution with 60 devices over an area of about 3.5×1.5 cm².However, increases in nanowire FET density, the use of cross-barcircuits and

implementing multiplexing/demultiplexing for addressing would allow thenanoES scaffolds to map cardiac and other synthetic tissue electricalactivities over the entire construct at higher densities in threedimensions.

FIG. 15A shows electrical recording traces from two devices in a cardiacpatch, before (left), during (middle) and after (right) norepinephrineapplication. The temporal difference between a pair of spikes from twodevices is denoted as delta-t_(N) (Δt_(N)). The interval betweenconsecutive spikes from a single device is denoted as t_(N)-t_(N-1),where N is the spike index. FIG. 15B shows a time (t) versus spike index(N) plot, showing a change in slope after norepinephrine application.The slopes correspond to the time-averaged <t_(N)-t_(N-1)>, and are 1.15seconds and 0.50 seconds before and after drug application,respectively. The data show that the cells exhibited overall coherentbeating and response to the drug. The right panel is a zoom-in view ofthe transition, where the middle point (N=23) shows a decreaseddelta-t_(N) (Δt_(N)) compared to earlier and later spikes. FIG. 15Cshows a Δt_(N) versus N plot. <Δt_(N)> and 1 SD (standard deviation)before (−) and after (+) norepinephrine application showed that althoughthe drug has minimum effect on <Δt_(N)>, the sub-millisecond andmillisecond fluctuations of delta-t_(N) (Δt_(N)) (1 SD) increased by ˜10fold following drug addition. Such stochastic variation demonstratesmillisecond-level, heterogeneous cellular responses to the drug.

The hybrid nanoES/neural 3D construct was prepared by culturing neuronswith a 3D reticular device array for 14 days in vitro with a densityof >4 million neurons/mL in Matrigel™. During recording, thenanoES/neural hybrid was perfused with an oxygenated artificial CSF(aCSF) containing (in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.3 MgSO₄, 1NaH₂PO₄, 26.2 NaHCO₃, 22 glucose and equilibrated with 95% O₂/5% CO₂.Three nanowire FETs (labeled 1, 2, and 3) were distributed in theconstruct with x-y-z positions shown in FIG. 16A. The total samplethickness was about 100 micrometers. The lines indicate the distancesbetween two devices in 3D. Sodium glutamate was dissolved in salinesolution and further diluted to 20 mM in aCSF solution. Glutamatesolution was injected in the middle above device 1 and 2 (arrow). Theinjection pulse duration is 0.5 s. FIG. 16B shows the local fieldpotential changes recorded from three devices in the 3D neuronconstruct, showing distinct position-dependent temporal responsesfollowing glutamate solution injection. FIG. 16C shows that perfusing6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) andD(−)-2-amino-5-phosphonopentanoic acid (APV) blockers prior to glutamateaddition eliminated any observed response, and thus showed that theobserved response in FIG. 16B could be attributed to postsynaptic signalpropagation. The segments above “Glutamate” mark the timing whenglutamate solution was injected (FIGS. 16B-16C)

Example 5

This example illustrates the development of artificial tissue withembedded nanoelectronic sensory capabilities. Vascular nanoES constructswere made by processes analogous to those used for tissue engineeredautologous blood vessels except for the addition of the nanoES (FIGS.17A and -18). In this example, human aortic smooth muscle cells (HASMCs)were cultured on 2D mesh nanoES with sodium ascorbate to promotedeposition of natural ECM. The hybrid sheets (FIG. 17B) were rolled intomulti-layer 3D tubular structures and matured (see below) withoutmacroscopic or desquamation (FIG. 17C). Cells in the construct expressedsmooth muscle alpha-actin (by immunostaining), the key contractileprotein in smooth muscle (FIG. 19).

The distribution of nanoES in the tubular construct was visualized bymicro-computed tomography (μCT). A top-down projection of reconstructed3D micro-computed tomography data (FIG. 17D) revealed regularly spacedmetal interconnects with at least four revolutions (arrows in FIG. 17D,Panel II), consistent with the NWFET mesh design and tissue rollingtechnique. Analyses of hematoxylineosin-stained sections (FIG. 17E)revealed well-defined smooth muscle tissue about 200 micrometers thick,with elongated cells and collagenous nanofibers, and embedded SU-8ribbons from the nanoES (FIG. 17E). These findings confirm 3Dintegration of NWFET nanoelectronics with healthy smooth muscle.

The potential of nanoES to function as a biomedical device, wasdemonstrated in the pH sensing capability of nanoES/HASMC vascularconstruct (FIG. 17F, inset). As the extravascular pH was varied stepwisewith luminal pH fixed, simultaneous recordings from NWFETs in theoutermost layer showed stepwise conductance decreases with a sensitivityof ˜32 mV/pH. NWFETs in the innermost layer (closest to luminal) showedminor baseline fluctuations. This ability to resolve extravascular pHchanges suggests the possibility of detecting inflammation, ischemia andtumor microenvironments or other forms of metabolic acidosis, e.g., dueto overproduction of organic acids or impaired renal acidification.Other markers of disease could potentially be detected by the nanoES.

Additional details regarding FIG. 17 follow. FIG. 17A shows a schematicof the synthesis of smooth muscle nanoES. The upper panels are sideviews, while the lower ones are either top views (Panels I and II) or azoom-in view (Panel III). The dots are NWFETs. Panel I shows a meshnanoES with NWFETs, while Panels II and III show the structure withHASMCs and collagenous matrix secreted by the HASMC. FIG. 17B, Panel Ishows a photograph of a single HASMC sheet cultured with sodiumL-ascorbate on a nanoES. Panel II shows a zoomed-in view of the dashedarea in Panel I, showing metallic interconnects macroscopicallyintegrated with cellular sheet. FIG. 17C is a photograph of the vascularconstruct after rolling into a tube and maturation in a culture chamberfor 3 weeks. FIG. 17D, Panel I is a microcomputed tomograph of a tubularconstruct segment. Panel II is a zoomed-in view of Panel I. Arrows markthe individual NWFET-containing layers of the rolled construct. Thescale bar is 1 mm. FIG. 6E shows hematoxylin & eosin (Panel I) andmasson trichrome (Panel II, with collagen) stained sections (about 6micrometers thick) cut perpendicular to the tube axis; lumen regions arelabeled. Thee arrows mark the positions of SU-8 ribbons of the nanoES.Scale bars are 50 micrometers. FIG. 17F shows changes in conductanceover time for two NWFET devices located in the outermost and innermostlayers. The inset is a schematic of the experimental set-up. Outertubing delivered bathing solutions with varying pH (dashed lines andarrows); inner tubing delivered solutions with fixed pH (dashed linesand arrows).

FIG. 19 is a confocal fluorescence microscopy image from the surface ofthe HASMC/mesh-like nanoelectronics biomaterial. In the image,alpha-actin in the smooth muscle cell was stained with Alexa Fluor® 488and the cell nuclei (round) were stained with Hoechst 34580. Localalignment of HASMCs is revealed by anisotropy in alpha-actin fibersrunning from upper left to lower right of image. The scale bars is 40micrometers.

Example 4

This example describes various methods used in Examples 1-3.

Methods Summary. Kinked and uniform silicon nanowires were synthesizedby nanocluster-catalyzed methods. See, e.g., U.S. Pat. No. 7,211,464,issued May 1, 2007, entitled “Doped Elongated Semiconductors, GrowingSuch Semiconductors, Devices Including Such Semiconductors, andFabricating Such Devices,” by Lieber, et al.; U.S. Pat. No. 7,301,199,issued Nov. 27, 2007, entitled “Nanoscale Wires and Related Devices,” byLieber, et al.; and International Patent Application No.PCT/US2010/050199, filed Sep. 24, 2010, entitled “Bent Nanowires andRelated Probing of Species,” by Tian, et al., published as WO2011/038228 on Mar. 31, 2011, each incorporated herein by reference inits entirety. The devices were fabricated on silicon substrates (NovaElectronic Materials, n-type 0.005 V cm) with 600 nm SiO₂ or 100SiO₂/200 Si₃N₄ at the surface. Electron beam lithography andphotolithography on nickel relief layers were used to define the metalcontacts to the nanowires and the key features of the scaffolds.

Steps used in the fabrication of the reticular nanoES included thefollowing. First, 100 nm nickel metal was patterned and deposited, andserved as the relief layer for the free-standing scaffolds. Next, a300-500 nm layer of SU-8 photoresist (2000.5, MicroChem, Newton) wasdeposited over the entire chip (FIG. 20C), followed by pre-baking at 65°C. and 95° C. for 2 and 4 min, respectively; then an isopropanolsolution of n⁺-n-n⁺ kinked nanowires was deposited onto the SU-8 layer.After identifying nanowire positions by optical imaging (Olympus BX51)and designing the interconnect and SU-8 patterns in IGOR Pro(WaveMetrics) and DesignCAD, EBL was used to pattern the overall SU-8scaffold structure around chosen nanowires, which was post-baked (65° C.and 95° C. for 2 and 4 min, respectively) and cured (180° C., 20 min) toyield the flexible structural support for metal interconnects. Thesilicon substrate was then coated with a methyl methacrylate andpoly(methyl methacrylate) double-layer resist, the resist was patternedover the chosen SU-8 ribbons and then non-symmetrical Cr/Pd/Cr(1.5/50-80/50-80 nm) metals were sequentially deposited followed bymetal lift-off in acetone to form the nanowire interconnects. Thenon-symmetrical Cr/Pd/Cr layer structure yielded a built-in stress,which drove 3D self-organization when the structure was relieved fromthe substrate.

The silicon substrate was then coated with a uniform 300-400 nm layer ofSU-8, and EBL of SU-8 followed by curing (180° C., 20 min) was used todefine the SU-8 passivation layer over the deposited metalinterconnects. The reticular nanoES, including the interconnected kinkednanowire FET devices, was released from the substrate by etching of thenickel layer (Nickel Etchant TFB, Transene Company, Danvers) for 60-120min at 25° C. Last, the free-standing nanoES was dried using a criticalpoint dryer (Autosamdri 815 Series A, Tousimis) and stored in a drystate before use in tissue culture. Each EBL step took about 10 min-2hours, depending on factors such as the writing speed and area, featuresize and complexity, and electron beam dosage (for example, the typicalarea dosages for SU-8 and poly(methyl methacrylate) EBL was 3-8 microCcm⁻² and 500-1,000 microC cm⁻² at 25 kV, respectively). The entirefabrication took 2-5 days, depending on the duration of the individualsteps. A similar approach was used in the fabrication of the mesh nanoESexcept that p-type nanowires and photolithography were used and theentire process took 2-3 days.

NanoES/collagen(Matrigel™) hybrid matrices were made by casting 50˜2,000microliters collagen or Matrigel™ solution onto the edge of (reticularnanoES) or directly above (mesh nanoES) the nanoES scaffolds, and at ˜4°C. The solutions were allowed to form gels around nanoES underconditions of 37° C. and 5% CO₂ for at least 20 min. The 3DnanoES/alginate scaffolds were prepared from pharmaceutical-gradealginate (Protanal LF5/60, FMC Biopolymers) by calcium gluconatecrosslinking and subsequent lyophilization to produce a sponge-likescaffold (5-15 mm×2-10 mm, d×h). To prepare NanoES/PLGA hybridscaffolds, a sheet of PLGA fibres with diameters of ˜1-3 micrometers wasdeposited on both sides of the mesh nanoES. The hybrid scaffold couldalso be folded to increase the thickness.

Embryonic Sprague/Dawley rat hippocampal cells, neonatal Sprague/Dawleyrat cardiomycytes and human aortic smooth muscle cells were cultured innanoES using established protocols. Optical micrographs ofimmunohistochemically and histologically stained samples were recordedusing either Olympus Fluoview FV1000 or Olympus FSX100 systems. Thestructures of nanoES were characterized with Zeiss Ultra55/Supra55VPfield-emission SEMs or the HMXST Micro-computed tomography x-ray imagingsystem (model: HMXST225, X-Tek). The in vitro cytotoxicity of nanoES wasevaluated using the standard LIVE/DEAD® Viability/Cytotoxicity Kit(Molecular Probes, Invitrogen) and the CellTiter 96® AQueous OneSolution Cell Proliferation Assay (Promega Corporation). Cardiomyocyterecordings were carried out in Tyrode solution with a 100 mV DC sourcefor the NWFETs. The current was amplified with a multi-channelpreamplifier, filtered with a 3 kHz low pass filter (CyberAmp 380), anddigitized at a 50 kHz sampling rate (Axon Digi1440A).

In extravascular pH sensing experiments, a single polydimethylsiloxane(PDMS) microfluidic chamber was used to deliver two flows of phosphatebuffer solutions, where inner and outer tubings were used to deliversolutions with fixed and varied pH, respectively. The electricalmeasurements were conducted using a lock-in amplifier with a modulationfrequency of 79 and 39 Hz, time constant of 30 ms, amplitude of 30 mV;the DC source-drain potential was zero. Ag/AgCl reference electrodeswere used in all recording and sensing experiments. The calibratedpotential values (in millivolts) recorded from nanowire FETs wereobtained as the ratios between device conductance changes (innanosiemens) and the sensitivities (in microsiemens per volt ornanosiemens per volt) that were determined individually in water-gateexperiments.

Nanowire Synthesis. Single-crystalline nanowires were synthesized usingthe Au nanoclustercatalyzed vapor-liquid-solid growth mechanism in ahome-built chemical vapor (CVD) deposition system. Au nanoclusters (TedPella Inc., Redding, Calif.) with either 20 or 80 nm diameters weredispersed on the oxide surface of silicon/SiO₂ substrates (600 nm oxide)and placed in the central region of a quartz tube CVD reactor system.Uniform 20 nm p-type silicon nanowires, which were used for mesh-likeNWFET scaffolds, were synthesized using established methods. See, e.g.,U.S. Pat. No. 7,211,464, issued May 1, 2007, entitled “Doped ElongatedSemiconductors, Growing Such Semiconductors, Devices Including SuchSemiconductors, and Fabricating Such Devices,” by Lieber, et al.; andU.S. Pat. No. 7,301,199, issued Nov. 27, 2007, entitled “Nanoscale Wiresand Related Devices,” by Lieber, et al., each incorporated herein byreference in its entirety. In a typical synthesis, the total pressurewas 40 torr and the flow rates of SiH₄, B₂H₆ (100 ppm in H₂) and H₂ were2, 2.5 and 60 standard cubic centimeters per minute (SCCM),respectively. The silicon-boron feed-in ratio was 4000:1, and the totalnanowire growth time was 30 min.

Kinked 80 nm diameter silicon nanowires, which were used for thereticular NWFET scaffolds, were synthesized with an⁺(arm)-n(device)-n⁺(arm) dopant profile. See, e.g., InternationalPatent Application No. PCT/US2010/050199, filed Sep. 24, 2010, entitled“Bent Nanowires and Related Probing of Species,” by Tian, et al.,published as WO 2011/038228 on Mar. 31, 2011, incorporated herein byreference in its entirety. In a typical synthesis, the total pressurewas 40 torr and the flow rates of SiH₄, PH₃ (1000 ppm in H₂) and H₂ were1, 5/0.1 and 60 sccm, respectively. Kinks were introduced by evacuationof the reactor (˜3×10⁻³ torr) for 15 s, and the silicon-phosphorusfeed-in ratios were 200:1 and 10,000:1 for the n⁺- and n-type segments,respectively. The n⁺-type arms were grown for 12-15 min, and the n-typeactive device channel segment was grown for 30 s immediately followingthe evacuation step used to introduce a kink.

Free-standing nanoES. The free-standing nanoES were fabricated on theoxide or nitride surfaces of silicon substrates (600 nm SiO₂ or 100SiO₂/200 Si₃N₄, n-type 0.005 V cm, Nova Electronic Materials, FlowerMound, Tex.) prior to relief from the substrate. Two basic types ofnanoES, termed reticular and mesh nanoES, were prepared. Componentsinclude silicon wafer (105 in FIG. 7), nickel relief layer (110),polymer ribbons (115), silicon nanowires (120), and metal interconnects(125). In a typical experiment, the widths of polymer and metalinterconnects were 1 and 0.7 micrometers, respectively. The built-instress from sequentially deposited Cr/Pd/Cr (1.5/50-80/50-80 nm) layersdrove self-organization into a 3D scaffold after the lift-off process.Key steps used in the fabrication of the reticular nanoES (FIG. 20) wereas follows: (1) Electron beam lithography (EBL) was used to pattern adouble layer resist of 500-600 nm of methyl methacrylate (MMA, MicroChemCorp., Newton, Mass.) and 100-200 nm of poly(methyl methacrylate) (PMMA,MicroChem Corp., Newton, Mass.), on which 100 nm nickel metal wasdeposited (FIGS. 20A-7B), where the nickel served as the final relieflayer for the free-standing scaffolds. (2) A 300-500 nm layer of SU-8photoresist (2000.5, MicroChem Corp., Newton, Mass.) was deposited overthe entire chip (FIG. 20C) followed by pre-baking at 65° C. and 95° C.for 2 and 4 min, respectively, then (3) an isopropanol solution ofn⁺n-n⁺ kinked nanowires was dropped onto the SU-8 layer and allowed toevaporate (FIG. 20D). (4) The kinked nanowire positions were locatedrelative to a standard marker patternS4 using an optical microscope(Olympus BX51) in dark-field mode, and then IGOR Pro (WaveMetrics) andDesignCAD were used to design the lithography patterns. EBL was thenused to pattern the overall SU-8 scaffold structure including a ringstructure underneath the selected kink nanowire. After post-baking (65°C. and 95° C. for 2 and 4 min, respectively), the SU-8 developer(MicroChem Corp., Newton, Mass.) was used to develop the SU-8 pattern.The areas exposed to electron beam became fully polymerized andinsoluble in SU-8 developer, which ‘glued’ the selected nanowires inplace. The rest of the SU-8 areas, including nanowires on theirsurfaces, were removed by SU-8 developer. After curing (180° C., 20min), patterned SU-8 ribbons as flexible structural support for metalinterconnects and nanowires were generated (FIG. 20E). (5) The siliconsubstrate was then coated with MMA and PMMA double layer resist, theresist was patterned over the chosen SU-8 ribbons, and thennonsymmetrical Cr/Pd/Cr (1.5/50-80/50-80 nm) metals were sequentiallydeposited followed by metal lift-off in acetone to form the nanowireinterconnects (FIG. 20F). The nonsymmetrical Cr/Pd/Cr layer structureyielded a built-in stress, which drove 3D self-organization when thestructure was relieved from the substrate. (6) The silicon substrate wasthen coated with a uniform 300-400 nm layer of SU-8, and EBL of SU-8followed by curing (180° C., 20 min) was used to define the SU-8passivation layer over the deposited metal interconnects (FIG. 20G). (7)The reticular nanoES, including the interconnected kinked NWFET devices,was released from the substrate by etching of the nickel layer (NickelEtchant TFB, Transene Company Inc., Danvers, Mass.) for 60-120 min at25° C. (FIG. 20H). Last, the free-standing nanoES were dried using acritical point dryer (Autosamdri 815 Series A, Tousimis, Rockville, Md.)and stored in the dry state prior to use in tissue culture. Here,self-organization produced random reticular scaffolds, but it should benoted that mechanics models and simulations (e.g., finite elementmethod) could be used to design and realize regular three-dimensional(3D) open framework constructs using the same approach.

A similar approach was used in the fabrication of the mesh nanoES (FIG.21): (1) Photolithography and metal deposition (100 nm, nickel) wereused to define a relief layer for the free-standing scaffold (FIGS. 21A,21B). (2) A layer of SU-8 photoresist (300-2000 nm, 2000.5 or 2002,MicroChem Corp., Newton, Mass.) was deposited over the entire chip, andphotolithography was used to pattern the bottom SU-8 mesh structure(FIG. 21C), which was then cured (180° C., 20 min). (3) A second 300-500nm thick layer of SU-8 was deposited over the entire chip, and prebakedat 65° C. and 95° C. for 2 and 4 min, respectively (FIG. 21D). (4) Thenp-type silicon nanowires were deposited from isopropanol solution andaligned by nitrogen blow-drying (FIG. 21E). (5) Photolithography andsubsequent curing (180° C., 20 min) was used to define the nanowirepatterns across the mesh structure and eliminate excess nanowires (FIG.21F). (6) The substrate was coated with S1805 and LOR 3A (MicroChemCorp., Newton, Mass.) double layer resist and patterned byphotolithography. Symmetrical Cr/Pd/Cr (1.5/50-100/1.5 nm) metals weresequentially deposited followed by metal lift-off in Remover PG(MicroChem Corp., Newton, Mass.) to define the minimally stressednanowire interconnects (FIG. 21G). (7) The substrate was coated with auniform layer of SU-8, and photolithography followed by curing (180° C.,20 min) was used to define a passivation layer over the deposited metalinterconnects (FIG. 21H). (8) The mesh-like nanoES was released from thesubstrate by etching the nickel layer (Nickel Etchant TFB, TranseneCompany Inc., Danvers, Mass.) for 6 h at 25° C. (FIG. 21I). Themesh-like scaffold adhered weakly to the substrate upon drying in air,and could be readily suspended as a freestanding scaffold upon immersionin water/cell culture medium. Scanning electron microscopy (SEM, ZeissUltra55/Supra55VP field-emission SEMs) was used to characterize bothtypes of fabricated scaffold structures.

NanoES/collagen(Matrigel™) hybrid matrix. Prior to gel casting, collagentype-I (Sigma-Aldrich Corp., St. Louis, Mo.) was diluted (1:2˜1:5) withculture media or phosphate buffered saline solution (PBS) and the pH wasadjusted to ˜7.4. Matrigel™ (BD Bioscience, Bedford, Mass.) was used asreceived or diluted (1:2˜1:5). Briefly, 50˜2000 microliters collagen orMatrigel solution was placed using a pipette (Eppendorf Research plus)onto the edge of (reticular nanoES) or directly above (mesh nanoES) thenanoES scaffolds, and at ˜4° C. The solutions were allowed to form gelsaround nanoES under 37° C., 5% CO₂ conditions for at least 20 min. Forvisualization of collagen fibers, fluorescein isothiocyanate labeledcollagen type-I (Sigma-Aldrich Corp., St. Louis, Mo.) was used.

NanoES/alginate hybrid scaffold. The 3D nanoES/alginate scaffolds wereprepared from pharmaceutical-grade alginate, Protanal LF5/60 (FMCBiopolymers), which has a high guluronic acid (G) content (65%).Briefly, (1) preparation of sodium alginate stock solutions atconcentrations of 1% (w/v); (2) partially crosslinking the alginatesolution by adding calcium gluconate; (3) drop casting partiallycrosslinked alginate onto loosely folded mesh nanoES, followed byadditional shaping and placement of nanoES inside the alginate gel witha glass rod; (4) freezing the nanoES/alginate gel in a homogeneous, cold(−20° C.) environment; and (5) lyophilization to produce a sponge likescaffold (5˜15 mm×2˜10 mm, d×h).

NanoES/PLGA hybrid scaffold. Poly(lactic-co-glycolic acid) (PLGA)electrospun fibers were used as a secondary scaffold in severalexperiments. The PLGA fibers were prepared based on typical proceduresas follows. PLGA (90/10 glycolide/L-lactide, inherent viscosity 1.71dL/g in HFIP at 25° C., Purac Biomaterials Inc.) was dissolved in1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma-Aldrich Corp., St. Louis,Mo.) at a 10 wt % concentration until a clear and homogenous solutionwas obtained. A syringe pump (Harvard Apparatus, Holliston, Mass.) wasused to deliver the polymer solution through a stainless steel capillaryat a rate of 3 mL/hr. A high voltage power supply (Gamma High VoltageResearch, Ormond Beach, Fla.) was used to apply a 25 kV potentialbetween the capillary tip and a grounded stainless steel plate 50 cmaway. Fibers were collected for 2-5 minutes before being put aside atroom temperature for 72 hours to allow residual solvent evaporate. Toprepare hybrid scaffolds, a sheet of PLGA fibers with diameters of ˜1-3micrometers was deposited on both sides of the mesh nanoES. The hybridscaffold could also be folded to increase its thickness.

Scaffold mechanical properties. The effective bending stiffness per unitwidth of the mesh scaffold, D, can be estimated by:

D=α _(s) D _(s)+α_(m) D _(m),

where α_(s) and α_(m) are the area fraction of the single-layer polymer(SU-8) ribbon (without metal layer and top polymer passivation layer)and three-layer ribbon (bottom polymer layer, metal layer and toppassivation layer) in the whole mesh structure. D_(s)=E_(z)h³/12 is thebending stiffness per unit width of the single-layer polymer, whereE_(z)=2 GPa and h are the modulus and thickness of the SU-8. D_(m) isthe bending stiffness per unit width of a three-layer structure, whichcan be calculated by:

${D_{m} = {\frac{E_{m}b_{m}h_{m}^{3}}{12b} + {\frac{E_{s}}{b}( {\frac{( {b - b_{m}} )( {{2h} + h_{m}} )^{3}}{12} + {\frac{1}{6}b_{m}h^{3}} + {2b_{m}{h( {\frac{h}{2} + \frac{h_{m}}{2}} )}^{2}}} )}}},$

where E_(m)=121 GPa and h_(m) are the modulus and thickness of thepalladium, b is the width of the single-layer ribbon and the total widthof the three-layer ribbon, b_(m) is the width of the palladium layer. Inaddition, the chromium layers are so thin (1.5 nm) that theircontribution to the bending stiffness is negligible. When h_(m)=75 nm,h=0.5 micrometers, b=10 micrometers, b_(m)=5 micrometers, α_(s)=2.51%and α_(m)=3.57%, D=0.006 nN m. When h_(m)=75 nanometers, h=2micrometers, b=40 micrometers, b_(m)=20 micrometers, α_(s)=10.06% andα_(m)=13.31%, D=1.312 nN m.

To calculate the strain in tubular constructs, the equation ε=y/R wasused, where y is the distance from the neutral plane, and R is theradius of curvature. For the symmetric mesh scaffold, since the neutralplane is the middle plane, the maximum strains of metal and SU-8 appearat Y=h_(m)/2 and y=h_(m)/2+h respectively. When h_(m)=75 nm, h=2micrometers, R=0.75 mm, the maximum strains of metal and SU-8 are 0.005%and 0.272%, respectively.

Scaffold structural simulation. The self-organization of the meshstructure due to residual stress was simulated by the commercial finiteelement software ABAQUS. Both the SU-8 ribbons and the SU-8/metalribbons were modeled as beam elements. The cross-sectional property ofthe SU-8/metal ribbons was defined by the appropriate meshed beamcross-section, while the cross-sectional property of the SU-8 ribbonswas set by defining the relevant rectangular profile. The equivalentbending moment on SU-8/metal ribbons was calculated using the residualstress measured by MET-1 FLX-2320-S thin film stress measurement system,which were 1.35 and 0.12 GPa for Cr (50 nm) and Pd (75 nm),respectively.

Neuron culture. Device chips were cleaned by oxygen plasma (50 sccm ofO₂, 50 w, 0.5 Torr, 1 min), and fixed onto a temperature controlledchamber (Warner Instruments, Hamden, Conn.) with double-sided tape (FIG.9A). A 1 mm thick polydimethylsiloxane (PDMS) membrane (Sylgard 184, DowCorning, Inc., Midland, Mich.) with 0.25 cm² open area in the center wascut, autoclaved and placed over the device area, followed bywire-bonding of individual devices. An autoclaved glass ring (ALAScientific Instruments, Farmingdale, N.Y.) was placed over this PDMSchamber and fixed with Kwik-Sil (World Precision Instruments, Inc.,Sarasota, Fla.) silicone elastomer (FIG. 9C). The whole chip wassterilized by UV illumination and 75% ethanol soak (20 min each). Anaqueous polylysine solution (0.5-1.0 mg/ml, MW 70,000 to 150,000,Sigma-Aldrich) was then introduced into the chamber and incubatedovernight at 37° C., the polylysine solution was removed, and thechamber rinsed 3 times each with 1× phosphate buffered saline (PBS)solution and NeuroPure Plating Medium (Genlantis, San Diego, Calif.).Finally, the chamber was filled with NeuroPure Plating Medium or culturemedium and conditioned in the incubator for 1 day. Hippocampal neurons(Gelantis, CA) were prepared using standard protocols. In brief, 5 mg ofNeuroPapain Enzyme (Gelantis, CA) was added to 1.5 ml of NeuroPrepMedium (Gelantis, San Diego, Calif.). The solution was kept at 37° C.for 15 min, and sterilized with a 0.2 micrometer syringe filter (PallCorporation, MI). Day 18 embryonic Sprague/Dawley rat hippocampal tissuewith shipping medium (E18 Primary Rat Hippocampal Cells, Gelantis, SanDiego, Calif.) was spun down at 200 g for 1 min. The shipping medium wasexchanged for NeuroPapain Enzyme medium. A tube containing tissue andthe digestion medium was kept at 30° C. for 30 min and manually swirledevery 2 min, the cells were spun down at 200 g for 1 min, theNeuroPapain medium was removed, and 1 ml of shipping medium was added.After trituration, cells were isolated by centrifugation at 200 g for 1min, then re-suspended in 5 to 10 mg/ml Matrigel™ (BD Bioscience,Bedford, Mass.) at 4° C. The cell/Matrigel™ mixture was plated on thereticular nanoES in the opening in the PDMS membrane at a density of 2to 4 million cells/ml and a total gel thickness of ˜0.5 to 1 mm. TheMatrigel™ matrix was allowed to gel at 37° C. for 20 min, then 1.5 ml ofNeuroPure Plating Medium was added, and the entire assembly was placedin the incubator. After 1 day, the plating medium was changed toNeurobasal™ medium (Invitrogen, Grand Island, N.Y.) supplemented withB27 (B27 Serum-Free Supplement, Invitrogen, Grand Island, N.Y.),Glutamax™ (Invitrogen, Grand Island, N.Y.) and 0.1% Gentamicin reagentsolution (Invitrogen, Grand Island, N.Y.). 3D neuron cultures weremaintained at 37° C. with 5% CO₂ for 7 to 21 days, with medium changedevery 4 to 6 days. For cultures lasting longer than 7 days,gas-permeable/water-impermeable membrane covers (ALA MEA-MEM-PL, ALAScientific Instruments, Farmingdale, N.Y.) were used to avoidevaporation while allowing for diffusion of gases (FIG. 9D).

Cardiomyocyte culture. Hybrid scaffolds (see FIG. 12B) of the meshnanoES (FIGS. 12A and 12G) sandwiched between two electrospun PLGA fiberlayers (1-3 micrometers diameter; 10-20 micrometers thick for individuallayer) were used in all experiments. The bottom PLGA layer was madeeither by inserting an existing layer underneath the mesh-like scaffoldor by directly electrospinning on the nanoES. The top PLGA layer wasmade by direct electrospinning on the nanoES.

The device chip was wire-bonded (FIGS. 12C and 12H), and assembled witha modified polystyrene petri-dish (VWR Inc.) using Kwik-Sil (WorldPrecision Instruments, Inc.) silicone elastomer glue (FIG. 12D). Thedevice chamber was cleaned by oxygen plasma (50 sccm of O₂, 50 w, 0.5Torr, 1 min), followed by sterilization with UV-light illumination for 1h and soaking in 70% ethanol solution for 0.5 h. The hybrid scaffoldswere coated with fibronectin/gelatin solution overnight prior to cellseeding. The fibronectin/gelatin solution was prepared by adding 0.1 gBacto-Gelatin (Fisher Scientific, DF0143-17-9) to 500 mL distilled waterin a glass bottle and autoclaving. The gelatin dissolved during theautoclaving step to yield a final concentration of gelatin of 0.02%. Oneml Fibronectin (Sigma, F-1141) was diluted in 199 ml of 0.02% gelatin.

Referring again to FIG. 12, FIG. 12A shows a freestanding mesh-likenanoES; FIG. 12B shows a hybrid of PLGA electrospun fibers and mesh-likenanoES; FIG. 12C shows that individual devices were wire-bonded to PCBconnecters; FIG. 12D shows that a modified petri dish was fixed over thescaffold with silicone elastomer; FIG. 12E shows that the hybridscaffold was sterilized by UV-light illumination for 1 h and soaking in70% ethanol solution for 0.5 h, coated with fibronectin/gelatin solutionovernight and seeded with cardiomyocytes/Matrigel™; and FIG. 12F showsthat after 1-2 days in culture, the cardiac sheet (from FIG. 12E) wasfolded and cultivated for an additional 3-10 days. FIG. 12G shows a meshdevice showing the free-standing part (the right half) and the fixedpart on the wafer (the left half). The arrow marks the outer-electrodepins for wire-bonding. FIG. 12H shows a printed circuit board (PCB) withwire-bonding wires. The wires connected the PCB copper pads (left) andthe rectangular electrodes on the supported end of the mesh-like nanoES(right). White dots highlight bonding points. Arrows highlight onewire-bonded aluminum wire.

Cardiac cells were isolated from intact ventricles of 1 to 3-day-oldneonatal Sprague/Dawley rats using 3 to 4 cycles (30 min each) of enzymedigestion using collagenase type II and pancreatin. The cells weresuspended in culture medium, composed of Medium-199 (Invitrogen, GrandIsland, N.Y.) supplemented with 0.6 mM CuSO₄.5H₂O, 0.5 mM ZnSO₄.7H₂O,1.5 mM vitamin B12, 500 U ml⁻¹ penicillin, 100 mg ml⁻¹ streptomycin and5 vol % fetal calf serum (FCS). The cardiac cells were finally seededwith 5-10 mg/ml Matrigel™ onto fibronectin/gelatin coated PLGA/meshnanoES at an initial cell density of 3-6×10⁷ cm⁻² (FIG. 12E). After 1-2days, the cell-seeded nanoES was manually folded into a construct, andwas maintained at 37° C. with 5% CO₂ for an additional 3-8 days (FIG.12F), with medium changes every 2-3 days. All animal proceduresconformed to US National Institutes of Health guidelines and wereapproved by Harvard University's Animal Care and Use Committee.

Vascular constructs. Synthetic vascular constructs were produced in amanner similar to the sheet-based tissue engineering approach describedpreviously (FIG. 18). First, the mesh nanoES were coated withgelatin/fibronectin solution overnight (FIGS. 18A-18C). Second, humanaortic smooth muscle cells (HASMC, Invitrogen, Grand Island, N.Y.) wereseeded at a density of 1×10⁴ cm⁻² on the gelatin/fibronectin-coateddevices and cultured in Medium 231 (Invitrogen, Grand Island, N.Y.)supplemented with smooth muscle growth supplement (SMGS, Invitrogen)(FIG. 14D). Sodium L-ascorbate (50 micrograms/mL, Sigma) was added tothe culture medium to stimulate extracellular matrix (ECM) synthesis.HASMCs were maintained at 37° C. with 5% CO₂ until their secreted ECMproteins formed an cohesive tissue sheet (7-14 days) that can be easilypeeled off from the silicon substrate. The cell-coated mesh nanoES wasthen gently lifted from the SiO₂ substrate using fine forceps, rolledonto a polystyrene or glass tubular support 1.5 mm in diameter, thenmaintained in culture Medium 231 supplemented with SMGS and 50micrograms/mL sodium L-ascorbate for at least another 2 weeks formaturation of the vascular structure (FIG. 18E).

0.5-2 h prior to pH sensing experiments, the temporary tubular supportwas removed, and segments of polystyrene tubing (the inner tubing inFIG. 17F, inset) were connected to the open ends of the vascularconstruct (FIG. 18F), and a PDMS fluidic chamber with input/outputtubing and Ag/AgCl electrodes (FIG. 17F, inset) was sealed with thesilicon substrate and the vascular construct using silicone elastomerglue (Kwik-Sil, World Precision Instruments, Inc.) as shown in FIG. 18H.Fresh medium was delivered to the vascular construct through both innerand outer tubing. The pH of the solution delivered through the outertubing was varied during the experiment.

FIG. 18A shows a free-standing mesh-like nanoES; FIG. 18B shows thatindividual devices were wirebonded to PCB connecters; FIG. 18C showsthat a modified petri-dish was fixed over the scaffold with siliconeelastomer; FIG. 18D shows that the hybrid scaffold was sterilized withUV-light illumination for 1 h and soaking in 70% ethanol solution for0.5 h, coated with fibronectin/gelatin solution overnight and seededwith HASMCs; FIG. 14E shows that after 7-14 days in culture, theHASMC-seeded nanoES (from FIG. 18D) was rolled against a tubular supportand cultivated for at least another 14 days; FIG. 18F shows that thetubular support was removed and tubing was connected to the ends of thelumen of the HASMC construct; FIG. 18G shows that the medium was removedwhile keeping the construct moist; and FIG. 18H shows that a PDMSchamber was assembled around the construct, attached to tubing to bathethe outside of the construct and Ag/AgCl electrodes to measure pH in thebathing fluid.

Immunochemical staining. Cells were fixed with 4% paraformaldehyde(Electron Microscope Sciences, Hatfield, Pa.) in PBS for 15-30 min,followed by 2-3 washes with ice-cold PBS. Cells were pre-blocked andpermeabilized (0.2-0.25% Triton X-100 and 10% feral bovine serum or 1%bovine serum albumin (BSA) in PBS) for 1 hour at room temperature. Next,the cells were incubated with primary antibodies in 1% BSA in 1×PBS with0.1% (v/v) Tween 20 (PBST) for 1 hr at room temperature or overnight at4° C. Then cells were incubated with the secondary antibodies withfluorophores. For counter-staining of cell nuclei, cells were incubatedwith 0.1-1 microgram/mL Hoechst 34580 (Molecular Probes, Invitrogen,Grand Island, N.Y.) for 1 min. Specific reagents used for different celltypes were as follows. Neurons: neuronal class III beta-Tubulin (TUJ1)mouse monoclonal antibody (1:500 dilution, Covance Inc., Princeton,N.J.) and AlexaFluor-546 goat anti-mouse IgG (1:1000, Invitrogen, GrandIsland, N.Y.) were used as the primary and secondary antibodies,respectively. Cardiomyocytes: anti-alpha-actinin mouse monoclonalantibody (1:450; Clone EA-53, Sigma-Aldrich Corp., St. Louis, Mo.) andAlexaFluor-488 goat anti-mouse (1:200; Molecular Probes, Invitrogen,Grand Island, N.Y.) were used as the primary and secondary antibodies,respectively. Hoechst 34580 was used to counter-stain cell nuclei.HASMC: anti-smooth muscle alpha-actin rabbit polyclonal antibody (1:500,Abcam, Cambridge, Mass.) and AlexaFluor-488 donkey anti-rabbit antibody(1:200; Molecular Probes, Invitrogen, Grand Island, N.Y.) were used asthe primary and secondary antibodies, respectively. Hoechst 34580 wasused to counter-stain cell nuclei.

Fluorescent dye labeling of devices and PLGA fibers. Fluorescence imagesof the reticular nanoES (FIGS. 4B and 6A) were obtained by doping theSU-8 resist solution with rhodamine 6G (Sigma-Aldrich Corp., St. Louis,Mo.) at a concentration less than 1 microgram/mL before deposition andpatterning. PLGA electrospun fiber scaffolds were labeled by physicalabsorption of rhodamine 6G from an aqueous solution (0.1 mg/mL), andthen rinsed copiously with water before fluorescence imaging.

Hematoxylin-eosin and Masson trichrome staining. The vascular constructswere cut and fixed in formalin solution (10%, neutral buffered,Sigma-Aldrich Corp., St. Louis, Mo.). The fixed sample was dehydrated ina series of graded ethanol baths (70% ethanol for 1 h, 95% ethanol for 1h, absolute ethanol 3× times, 1 h each) and xylenes (2×, 1 h each), andthen infiltrated with molten paraffin (HistoStar, Thermo Scientific,Kalamazoo, Mich.) at 58° C. for 2 h. The infiltrated tissues wereembedded into paraffin blocks and cut into 5-6 micrometer sections.Immediately prior to straining, the paraffin was removed from thesections by 2 washes with xylene, 1 min each. Then the sections wererehydrated by a 5 min wash in absolute ethanol, 2 min in 95% ethanol, 2min in 70% ethanol and 5 min in distilled water. Standard hematoxylinand eosin staining was carried out using an automated slide stainer(Varistain Gemini ES, Thermo Scientific, Kalamazoo, Mich.). Collagensecretion by HASMCs was assessed on deparaffinized sections using aMasson's trichrome staining kit (Polysciences, Inc., Warrington, Pa.)according to standard protocols.

Optical microscopy and image analysis. Confocal and epi-fluorescenceimaging were carried out using an Olympus Fluoview FV1000 confocal laserscanning microscope. Confocal images were acquired using 405, 473 and559 nm wavelength lasers to excite cellular components labeled withHoechst 34580, AlexaFluor-488/Rodamine-6G, andRodamine-6G/AlexaFluor-546 fluorescent dyes (Molecular Probes andSigma-Aldrich Corp.), respectively. A 635 nm wavelength laser was usedfor imaging metal interconnects in reflective mode. Epi-fluorescenceimages were acquired using a mercury lamp together with standard DAPI(EX:377/50,EM:447/60), GFP (EX:473/31,EM520/35) and TRITC(EX:525/40,EM:585/40) filters. ImageJ (ver. 1.45i, Wayne Rasband,National Institutes of Health, USA) was used for 3D reconstruction andanalysis of the confocal and epi-fluorescence images. Bright-fieldoptical micrographs of histological samples were acquired on an OlympusFSX100 system using FSXBSW software (ver. 02.02).

Micro-computed tomography. The nanoES in the synthetic vascularconstruct was imaged using a HMXST Micro-CT x-ray imaging system with astandard horizontal imaging axis cabinet (model:HMXST225, NikonMetrology, Inc., Brighton, Mich.). Prior to imaging, samples were fixedand dried. In a typical imaging process, 60-70 kV acceleration voltageand 130-150 microamperes electron beam current was used. No filter wasused. VGStudio MAX (ver. 2.0, Volume Graphics GMbh, Germany) was usedfor 3D reconstruction and analysis of the micro-CT images.

Cell viability assays. Hippocampal neuron viability was evaluated usinga LIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes, Invitrogen,Grand Island, N.Y.). On days 7, 14 and 21 of the culture, neurons wereincubated with 1 micromolar calcein-AM and 2 micromolar ethidiumhomodimer-1 (EthD-1) for 45 min at 37° C. to label live and dead cells,respectively. Cell viability at each time point was calculated aslive/(live+dead)×100, and was normalized to the percentage of live cellson day 0 (live_(day n)/live_(day 0)). Three-dimensional neuron culturesin Matrigel™ on polylysine modified glass slides (Fisher ScientificInc., Waltham, Mass.) were used as controls. The cells were imaged witha confocal fluorescence microscope (Olympus Fluoview FV1000) and the 3Dreconstructed images were used for live/dead cell counting. For eachgroup, n=6. In 3D cardiac cultures, cell viability was evaluated with anassay of a mitochondrial metabolic activity, the CellTiter 96® AQueousOne Solution Cell Proliferation Assay (Promega Corp., Madison, Wis.)that uses a tetrazolium compound(3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS) and an electron coupling reagent (phenazineethosulfate; PES). On days 2, 4, 6, 8, 10 and 12 of the culture, cardiacconstructs were incubated with CellTiter 96® AQueous One Solution for120 min at 37° C. The absorbance of the culture medium at 490 nm wasimmediately recorded with a 96-well plate reader. The quantity offormazan product (converted from tetrazole) as measured by theabsorbance at 490 nm was directly proportional to cell metabolicactivity in culture. Three-dimensional cardiomyocyte cultures inMatrigel™ on gelatin coated electrospun PLGA fibers were used ascontrols. For each group, n=6.

Electrical measurements. The nanowire FET conductance andtransconductance (sensitivity) were measured in 1×PBS. The slope of alinear fit to conductance versus water-gate potential (V_(gate)) datawas used to determine transconductance. For NWFET stability tests, thereticular NWFET devices were maintained under neuron culture conditions(see details above, in Neuron culture) for predetermined intervals.Electrical transport measurements and recordings from 3Dcardiomyocyte-seeded nanoES were obtained in Tyrode solution (pH ˜7.3)with a 100 mV DC source voltage at 25° C. or 37° C. The current wasamplified with a multi-channel current/voltage preamplifier, filteredwith a 3 kHz low pass filter (CyberAmp 380), and digitized at a 50 kHzsampling rate (Axon Digi1440A). In extravascular pH sensing experiments,a single polydimethylsiloxane (PDMS) microfluidic chamber was used todeliver two flows of phosphate buffer solutions: the pH delivered by theouter input tubing was varied, while that of the inner tubing was fixedat 7.4. In the pH-sensing experiments, nanoelectronic devices weremodulated using a lock-in amplifier with a modulation frequency of 79and 39 Hz, time constant of 30 ms, amplitude of 30 mV, and DCsource-drain potential of zero. Ag/AgCl reference electrodes were usedin all recording and sensing experiments.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is: 1-47. (canceled)
 48. A method, comprising: formingone or more polymeric constructs on a substrate using e-beamlithography, wherein the polymeric constructs comprise one or morenanoscale wires; removing at least a portion the substrate from the oneor more polymeric constructs; and rolling the one or more polymericconstructs into a 3-dimensional structure.
 49. The method of claim 48,wherein the substrate comprises a sacrificial material.
 50. The methodof claim 49, wherein the sacrificial material comprises nickel.
 51. Themethod of claim 49, further comprising depositing the sacrificialmaterial on the substrate, prior to forming the one or more polymericconstructs on a substrate.
 52. The method of claim 51, wherein formingthe one or more polymeric constructs on the substrate comprises:depositing one or more bedding polymers on at least a portion of thesacrificial material as one or more layers; and depositing one or morenanoscale wires on at least a portion of the uppermost layer of beddingpolymer.
 53. The method of claim 52, wherein at least one of the beddingpolymers comprises a photoresist.
 54. The method of claim 52,comprising: depositing a first bedding polymer on at least a portion ofthe sacrificial material; depositing a second bedding polymer on atleast a portion of the first bedding polymer; and depositing one or morenanoscale wires on at least a portion of the second bedding polymer. 55.The method of claim 52, further comprising aligning the depositednanoscale wires.
 56. The method of claim 49, further comprising removingat least a portion of the sacrificial material.
 57. The method of claim56, wherein upon removing at least a portion of the sacrificialmaterial, the one or more polymeric constructs spontaneously rolls intothe 3-dimensional structure.
 58. The method of claim 48, furthercomprising forming one or more metal leads on the polymeric material.59. The method of claim 58, wherein forming one or more metal leadscomprises: depositing one or more lead polymers on at least some of theone or more the nanoscale wires; depositing one or more metals on atleast a portion of the one or more lead polymers; and removing a portionof the one or more metals to form the one or more metal leads.
 60. Themethod of claim 59, wherein depositing one or more metals on at least aportion of the one or more lead polymers comprises: depositing a firstmetal on the one or more lead polymers; and depositing a second metal onat least a portion of the first metal.
 61. The method of claim 60,further comprising depositing a third metal on at least a portion of thesecond metal.
 62. The method of claim 61, wherein the first metal, thesecond metal, and the third metal are deposited in a pre-stressedarrangement.
 63. The method of claim 58, further comprising connectingthe one or more metal leads to an external electrical circuit.
 64. Themethod of claim 48, wherein the one or more polymeric constructscomprises a biocompatible polymer layer.
 65. The method of claim 48,further comprising depositing an extracellular matrix protein on atleast a portion of the one or more one or more polymeric constructs. 66.The method of claim 48, further comprising seeding one or more cellsonto the one or more one or more polymeric constructs.
 67. The method ofclaim 48, further comprising exposing the one or more polymericconstructs to a cell growth medium.